Monomethylvaline compounds having phenylalanine side-chain replacements at the C-terminus

ABSTRACT

Auristatin peptide analogs of MeVal-Val-Dil-Dap-Phe (MMAF) are provided having C-terminal phenylalanine residue side chain replacements or modifications which are provided alone or attached to ligands through various linkers. The related conjugates can target specific cell types to provide therapeutic benefit.

CONTINUITY

This application is the U.S. National Phase entry under 35 U.S.C. §371of International Patent Application No. PCT/US06/26352, filed Jul. 7,2006 which claims priority from and the benefit of U.S. ProvisionalApplication No. 60/697,767, filed Jul. 7, 2005; the disclosures of whichare incorporated by reference herein.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file-15-1.TXT, created on Mar. 8, 2012,4,096 bytes, machine format IBM-PC, MS-Windows operating system, ishereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

The present invention is directed to Drug Compounds, toDrug-Linker-Ligand Conjugates, Drug-Linker Compounds, and Drug-LigandConjugates; as well as to compositions including the same, and tomethods for using the same to treat cancer, an autoimmune disease, aninfectious disease and other pathological conditions. The invention alsorelates to methods of using Antibody-Drug Conjugate compounds in vitro,in situ, and in vivo for the diagnosis or treatment of mammalian cells,or associated pathological conditions.

BACKGROUND OF THE INVENTION

A great deal of interest has surrounded the use of monoclonal antibodies(mAbs) for the selective delivery of cytotoxic agents to tumor cells.MMAF (N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine) isan auristatin that is relatively non-toxic, yet is highly potent inactivity when conjugated to internalizing mAbs. MMAF has a chargedC-terminal phenylalanine residue that attenuates its cytotoxic activitycompared to its neutral counterpart, MMAE; this difference is mostlikely due to impaired intracellular access. However, conjugating MMAFto internalizing antibodies, like AC10 or 1F6, via a protease cleavablelinker resulted in conjugates that are >2000 fold more potent on antigenpositive cells as compared to unconjugated drug. Active targeting withmAbs facilitates intracellular delivery of MMAF; once MMAF is releasedfrom the conjugate inside cells the drug, it is presumably trapped dueto its reduced ability to cross cellular membranes thus increasing itsintracellular concentration and therefore the potency of the conjugate.Using cytotoxic drugs with impaired passive intracellular uptake canpotentially lead to mAb-drug conjugates with reduced systemic toxicity.Indeed, non-specific cleavage of the linker in circulation would releasea relatively non-toxic drug.

To expand and improve the auristatin class of drugs, and thecorresponding antibody drug conjugates (ADCs), the side chain of theC-terminal phenylalanine residue of MMAF has been modified. Thisstructural modification imparts unexpected properties to the resultantfree drug and ADC.

The recitation of any reference in this application is not an admissionthat the reference is prior art to this application.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides compounds and conjugatesrepresented by the general formulaL_(v)-[(LU)₀₋₁-(D)₁₋₄]_(p)wherein L is H or a Ligand unit; LU is a Linker unit; v is 0 or 1; p isan integer of from 1 to 20; and D is a drug moiety having the formula:

wherein R² is selected from H and C₁-C₈ alkyl; R³ is selected from H,C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, X¹-aryl, X¹—(C₃-C₈ carbocycle),C₃-C₈ heterocycle and X¹—(C₃-C₈ heterocycle); R⁴ is selected from H,C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, X¹-aryl, X¹—(C₃-C₈ carbocycle),C₃-C₈ heterocycle and X¹—(C₃-C₈ heterocycle); R⁵ is selected from H andmethyl; or R⁴ and R⁵ jointly form a carbocyclic ring and have theformula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independentlyselected from H, C₁-C₈ alkyl and C₃-C₈ carbocycle and n is selected from2, 3, 4, 5 and 6; R⁶ is selected from H and C₁-C₈ alkyl; R⁷ is selectedfrom H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, X¹-aryl, X¹—(C₃-C₈carbocycle), C₃-C₈ heterocycle and X¹—(C₃-C₈ heterocycle); each R⁸ isindependently selected from H, OH, C₁-C₈ alkyl, C₃-C₈ carbocycle andO—(C₁-C₈ alkyl); wherein each X¹ is a C₁-C₁₀ alkylene and the moiety—NR⁹Z¹ is a phenylalanine bioisostere with a modified amino acid sidechain; or a pharmaceutically acceptable salt or solvate thereof.

The compounds of the above formulas are useful for treating disorders,such as cancer, autoimmune disease or infectious disease, in a patientor useful as an intermediate for the synthesis of a Drug-Linker Compoundor a Drug-Linker-Ligand Conjugate (e.g., a Drug-Linker-AntibodyConjugate, a Drug-Ligand Conjugate, or a Drug-Ligand Conjugate having acleavable Drug unit).

In another aspect, compositions are provided that include an effectiveamount of a compound of the above formulae and a pharmaceuticallyacceptable carrier or vehicle.

In yet another aspect, methods for killing or inhibiting themultiplication of a tumor cell or cancer cell are provided. In stillanother aspect, methods for treating cancer are provided. In stillanother aspect, methods for killing or inhibiting the replication of acell that expresses an autoimmune antibody are provided. In yet anotheraspect, methods for treating an autoimmune disease are provided. Instill another aspect, methods for treating an infectious disease areprovided. In yet another aspect, methods for preventing themultiplication of a tumor cell or cancer cell are provided. In stillanother aspect, methods for preventing cancer are provided. In stillanother aspect, methods for preventing the multiplication of a cell thatexpresses an autoimmune antibody are provided. In yet another aspect,methods for preventing an autoimmune disease are provided. In stillanother aspect, methods for preventing an infectious disease areprovided.

In another aspect, a Drug Compound is provided that can be used as anintermediate for the synthesis of a Drug-Linker Compound having acleavable Drug unit.

In another aspect, a Drug-Linker Compound is provided that can be usedas an intermediate for the synthesis of a Drug-Linker-Ligand Conjugate.

In another aspect, an assay is provided for detecting cancer cells, theassay including:

(a) exposing the cells to an Antibody Drug Conjugate compound; and

(b) determining the extent of binding of the Antibody Drug Conjugatecompound to the cells.

The invention will best be understood by reference to the followingdetailed description of the exemplary embodiments, taken in conjunctionwith the accompanying drawings, figures, and schemes. The discussionbelow is descriptive, illustrative and exemplary and is not to be takenas limiting the scope defined by any appended claims.

DETAILED DESCRIPTION OF THE INVENTION

Definitions and Abbreviations

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings. When trade names are usedherein, the trade name includes the product formulation, the genericdrug, and the active pharmaceutical ingredient(s) of the trade nameproduct, unless otherwise indicated by context.

The term “antibody” herein is used in the broadest sense andspecifically covers intact monoclonal antibodies, polyclonal antibodies,monospecific antibodies, multispecific antibodies (e.g., bispecificantibodies) formed from at least two intact antibodies, and antibodyfragments, that exhibit the desired biological activity. An intactantibody has primarily two regions: a variable region and a constantregion. The variable region binds to and interacts with a targetantigen. The variable region includes a complementary determining region(CDR) that recognizes and binds to a specific binding site on aparticular antigen. The constant region may be recognized by andinteract with the immune system (see, e.g., Janeway et al., 2001,Immuno. Biology, 5th Ed., Garland Publishing, New York). An antibody canbe of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. The antibody can bederived from any suitable species. In one aspect, however, the antibodyis of human, murine, or rabbit origin. An antibody can be, for example,human, humanized or chimeric, a single chain antibody, an Fv fragment,an Fab fragment, an F(ab′) fragment, an F(ab′)₂ fragment, a fragment(s)produced by a Fab expression library, anti-idiotypic (anti-Id)antibodies, and epitope-binding fragments of any of the above whichimmunospecifically bind to a target antigen (e.g., a cancer cellantigen, a viral antigen or a microbial antigen).

The terms “specifically binds” and “specific binding” refer to antibodybinding to a predetermined antigen. Typically, the antibody binds withan affinity of at least about 1×10⁷ M⁻¹, and binds to the predeterminedantigen with an affinity that is at least two-fold greater than itsaffinity for binding to a non-specific antigen (e.g., BSA, casein) otherthan the predetermined antigen or a closely-related antigen.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. The modifier “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method.

The term “monoclonal antibodies” specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen-binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments, diabodies, triabodies, tetrabodies, linear antibodies,single-chain antibody molecules, scFv, scFv-Fc, and multispecificantibodies formed from antibody fragment(s).

An “intact” antibody is one which comprises an antigen-binding variableregion as well as a light chain constant domain (C_(L)) and heavy chainconstant domains, C_(H)1, C_(H)2, C_(H)3 and C_(H)4, as appropriate forthe antibody class. The constant domains may be native sequence constantdomains (e.g., human native sequence constant domains) or amino acidsequence variant thereof.

An intact antibody may have one or more “effector functions” whichrefers to those biological activities attributable to the Fc region(e.g., a native sequence Fc region or amino acid sequence variant Fcregion) of an antibody. Examples of antibody effector functions includeC1q binding; complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc.

A “native sequence” polypeptide is one which has the same amino acidsequence as a polypeptide, e.g., a tumor-associated antigen receptor,derived from nature. Such native sequence polypeptides can be isolatedfrom nature or can be produced by recombinant or synthetic means. Thus,a native sequence polypeptide can have the amino acid sequence of anaturally-occurring human polypeptide, a murine polypeptide, or apolypeptide from any other mammalian species.

The term “amino acid sequence variant” refers to polypeptides havingamino acid sequences that differ to some extent from a native sequencepolypeptide. Ordinarily, amino acid sequence variants will possess atleast about 70% homology with at least one receptor binding domain of anative ligand, or with at least one ligand binding domain of a nativereceptor, such as a tumor-associated antigen. In other aspects, theywill be at least about 80%, at least about 90%, or at least 95%homologous with such receptor or ligand binding domains. The amino acidsequence variants possess substitutions, deletions, and/or insertions atcertain positions within the amino acid sequence of the native aminoacid sequence.

“Sequence identity” is defined as the percentage of residues in theamino acid sequence variant that are identical after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. A preferred, non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl.Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into theNBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol.215:403-410. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleic acid encoding a protein of interest. BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3, to obtain amino acid sequences homologous to protein ofinterest. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., 1997, NucleicAcids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to performan iterated search which detects distant relationships between molecules(Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. Another preferred, non-limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller, CABIOS (1989). Such an algorithm is incorporated intothe ALIGN program (version 2.0) which is part of the GCG sequencealignment software package. When utilizing the ALIGN program forcomparing amino acid sequences, a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4 can be used. Additionalalgorithms for sequence analysis are known in the art and includeADVANCE and ADAM as described in Torellis and Robotti, 1994, Comput.Appl. Biosci. 10:3-5; and FASTA described in Pearson and Lipman, 1988,Proc. Natl. Acad. Sci. USA 85:2444-8. Within FASTA, ktup is a controloption that sets the sensitivity and speed of the search. If ktup=2,similar regions in the two sequences being compared are found by lookingat pairs of aligned residues; if ktup=1, single aligned amino acids areexamined. ktup can be set to 2 or 1 for protein sequences, or from 1 to6 for DNA sequences. The default if ktup is not specified is 2 forproteins and 6 for DNA. Alternatively, protein sequence alignment may becarried out using the CLUSTAL W algorithm, as described by Higgins etal., 1996, Methods Enzymol. 266:383-402. In some embodiments, the twosequences that are compared are the same length after gaps areintroduced within the sequences, as appropriate (e.g., excludingadditional sequence extending beyond the sequences being compared).

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells in summarized is Table 3on page 464 of Ravetch and Kinet, 1991, Annu. Rev. Immunol. 9:457-92. Toassess ADCC activity of a molecule of interest, an in vitro ADCC assay,such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may beperformed. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al., 1998, Proc. Natl. Acad. Sci. USA 95:652-656.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and Fcγ RIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), that havesimilar amino acid sequences that differ primarily in the cytoplasmicdomains thereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (See review M. inDaëron, 1997, Annu. Rev. Immunol. 15:203-234). FcRs are reviewed inRavetch and Kinet, 1991, Annu. Rev. Immunol., 9:457-92; Capel et al.,1994, Immunomethods 4:25-34 (1994); and de Haas et al., 1995, J. Lab.Clin. Med. 126:330-41. Other FcRs, including those to be identified inthe future, are encompassed by the term “FcR” herein. The term alsoincludes the neonatal receptor, FcRn, which is responsible for thetransfer of maternal IgGs to the fetus. (See, e.g., Guyer et al., 1976,J. Immunol. 117:587; and Kim et al., 1994, J. Immunol. 24:249).

“Complement dependent cytotoxicity” or “CDC” refers to the ability of amolecule to lyse a target in the presence of complement. The complementactivation pathway is initiated by the binding of the first component ofthe complement system (C1q) to a molecule (e.g., an antibody) complexedwith a cognate antigen. To assess complement activation, a CDC assay,e.g., as described in Gazzano-Santoro et al., 1996, J. Immunol. Methods202:163, may be performed.

The term “variable” refers to certain portions of the variable domainsof antibodies that differ extensively in sequence and are used in thebinding and specificity of each particular antibody for its particularantigen. This variability is concentrated in three segments called“hypervariable regions” in the light chain and the heavy chain variabledomains. The more highly conserved portions of variable domains arecalled the framework regions (FRs). The variable domains of native heavyand light chains each comprise four FRs connected by three hypervariableregions.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g., residues 24-34(L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variabledomain; Kabat et al. (Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md. (1991)) and/or those residues from a “hypervariable loop” (e.g.,residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavychain variable domain; Chothia and Lesk, 1987, J. Mol. Biol.196:901-917). “Framework Region” or “FR” residues are those variabledomain residues other than the hypervariable region residues as hereindefined.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Typically, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thescFv to form the desired structure for antigen binding. For a review ofscFv, see Plütckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a variable heavy domain(V_(H)) connected to a variable light domain (V_(L)) in the samepolypeptide chain. By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. Diabodies are described more fully in, forexample, EP 0 404 097; WO 93/11161; and Hollinger et al., 1993, Proc.Natl. Acad. Sci. USA 90:6444-6448.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., 1986, Nature321:522-525; Riechmann et al., 1988, Nature 332:323-329; and Presta,1992, Curr. Op. Struct. Biol. 2:593-596.

As used herein, “isolated” means separated from other components of (a)a natural source, such as a plant or animal cell or cell culture, or (b)a synthetic organic chemical reaction mixture. As used herein,“purified” means that when isolated, the isolate contains at least 95%,and in another aspect at least 98%, of a compound (e.g., a conjugate) byweight of the isolate.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An antibody “which binds” an antigen of interest is one capable ofbinding that antigen with sufficient affinity such that the antibody isuseful in targeting a cell expressing the antigen.

An antibody which “induces apoptosis” is one which induces programmedcell death as determined by binding of annexin V, fragmentation of DNA,cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation,and/or formation of membrane vesicles (called apoptotic bodies). Thecell is a tumor cell, e.g., a breast, ovarian, stomach, endometrial,salivary gland, lung, kidney, colon, thyroid, pancreatic or bladdercell. Various methods are available for evaluating the cellular eventsassociated with apoptosis. For example, phosphatidyl serine (PS)translocation can be measured by annexin binding; DNA fragmentation canbe evaluated through DNA laddering; and nuclear/chromatin condensationalong with DNA fragmentation can be evaluated by any increase inhypodiploid cells.

The term “therapeutically effective amount” refers to an amount of adrug effective to treat a disease or disorder in a mammal. In the caseof cancer, the therapeutically effective amount of the drug may reducethe number of cancer cells; reduce the tumor size; inhibit (i.e., slowto some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy,efficacy can, for example, be measured by assessing the time to diseaseprogression (TTP) and/or determining the response rate (RR).

The term “substantial amount” refers to a majority, i.e. >50% of apopulation, of a collection or a sample.

The term “intracellular metabolite” refers to a compound resulting froma metabolic process or reaction inside a cell on a Drug-Linker-Ligandconjugate (e.g., an antibody drug conjugate (ADC)). The metabolicprocess or reaction may be an enzymatic process such as proteolyticcleavage of a peptide linker of the ADC, by hydrolysis of a functionalgroup such as a hydrazone, ester, or amide, or by proteolyticdegradation of the Drug-Linker-Ligand conjugate (e.g., releasing acystyl-Linker-Drug fragment). Intracellular metabolites include, but arenot limited to, antibodies and free drug which have undergoneintracellular cleavage after entry, diffusion, uptake or transport intoa cell.

The terms “intracellularly cleaved” and “intracellular cleavage” referto a metabolic process or reaction inside a cell on an Drug-LigandConjugate, a Drug-Linker-Ligand Conjugate, an Antibody Drug Conjugate(ADC) or the like, whereby the covalent attachment, e.g., the linker,between the drug moiety (D) and the antibody (Ab) is broken, resultingin the free Drug, a Drug-Linker Compound or other metabolite of theConjugate dissociated from the antibody inside the cell. The cleavedmoieties of the Drug-Ligand Conjugate, a Drug-Linker-Ligand Conjugate orADC are thus intracellular metabolites.

The term “bioavailability” refers to the systemic availability (i.e.,blood/plasma levels) of a given amount of drug administered to apatient. Bioavailability is an absolute term that indicates measurementof both the time (rate) and total amount (extent) of drug that reachesthe general circulation from an administered dosage form.

The term “cytotoxic activity” refers to a cell-killing, cytostatic oranti-proliferation effect of an antibody drug conjugate compound or anintracellular metabolite of an antibody drug conjugate compound.Cytotoxic activity may be expressed as the IC₅₀ value which is theconcentration (molar or mass) per unit volume at which half the cellssurvive.

A “disorder” is any condition that would benefit from treatment. Thisincludes chronic and acute disorders or diseases including thosepathological conditions which predispose the mammal to the disorder inquestion. Non-limiting examples of disorders to be treated hereininclude benign and malignant tumors; leukemia and lymphoid malignancies,in particular breast, ovarian, stomach, endometrial, salivary gland,lung, kidney, colon, thyroid, pancreatic, prostate or bladder cancer;neuronal, glial, astrocytal, hypothalamic and other glandular,macrophagal, epithelial, stromal and blastocoelic disorders; andinflammatory, angiogenic and immunologic disorders.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition or disorder in mammals that is typicallycharacterized by unregulated cell growth. A “tumor” comprises one ormore cancerous cells. Examples of cancer include, but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers include squamouscell cancer (e.g., epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer, non-small cell lung cancer (“NSCLC”),adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, rectal cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, anal carcinoma, penile carcinoma, as well as head and neckcancer.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, ⁶⁰C, andradioactive isotopes of Lu), chemotherapeutic agents, and toxins such assmall molecule toxins or enzymatically active toxins of bacterial,fungal, plant or animal origin, including synthetic analogs andderivatives thereof. In one aspect, the term does not include aradioactive isotope(s).

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, and IL-12; a tumor necrosis factor suchas TNF-α or TNF-β; and other polypeptide factors including LIF and kitligand (KL). As used herein, the term cytokine includes proteins fromnatural sources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically or hydrolytically activated or converted into themore active parent form. See, e.g., Wilman, “Prodrugs in CancerChemotherapy” Biochemical Society Transactions, 14, pp. 375-382, 615thMeeting Belfast (1986) and Stella et al., “Prodrugs: A Chemical Approachto Targeted Drug Delivery,” Directed Drug Delivery, Borchardt et al.,(ed.), pp. 247-267, Humana Press (1985). The prodrugs of this inventioninclude, but are not limited to, phosphate-containing prodrugs,thiophosphate-containing prodrugs, sulfate-containing prodrugs,peptide-containing prodrugs, D-amino acid-modified prodrugs,glycosylated prodrugs, β-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs or optionallysubstituted phenylacetamide-containing prodrugs, 5-fluorocytosine andother 5-fluorouridine prodrugs which can be converted into the moreactive cytotoxic free drug. Examples of cytotoxic drugs that can bederivatized into a prodrug form for use in this invention include, butare not limited to, those chemotherapeutic agents described above.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as, for example, anti-CD20, CD30, CD33, CD40, CD70, BCMA, or LewisY antibodies and, optionally, a chemotherapeutic agent) to a mammal. Thecomponents of the liposome are commonly arranged in a bilayer formation,similar to the lipid arrangement of biological membranes.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indication(s), usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe nucleic acid. An isolated nucleic acid molecule is other than in theform or setting in which it is found in nature. Isolated nucleic acidmolecules therefore are distinguished from the nucleic acid molecule asit exists in natural cells. However, an isolated nucleic acid moleculeincludes a nucleic acid molecule contained in cells that ordinarilyexpress the nucleic acid where, for example, the nucleic acid moleculeis in a chromosomal location different from that of natural cells.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA encoding apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence, for example, if it affects the transcription ofthe sequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingcan be accomplished by ligation at convenient restriction sites. If suchsites do not exist, the synthetic oligonucleotide adaptors or linkerscan be used in accordance with conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

An “autoimmune disease” herein is a disease or disorder arising from anddirected against an individual's own tissues or a co-segregate ormanifestation thereof or resulting condition therefrom. Examples ofautoimmune diseases or disorders include, but are not limited toarthritis (rheumatoid arthritis, juvenile rheumatoid arthritis,osteoarthritis, psoriatic arthritis, and ankylosing spondylitis),psoriasis, dermatitis including atopic dermatitis; chronic idiopathicurticaria, including chronic autoimmune urticaria,polymyositis/dermatomyositis, toxic epidermal necrolysis, systemicscleroderma and sclerosis, responses associated with inflammatory boweldisease (IBD) (Crohn's disease, ulcerative colitis), and IBD withco-segregate of pyoderma gangrenosum, erythema nodosum, primarysclerosing cholangitis, and/or episcleritis), respiratory distresssyndrome, including adult respiratory distress syndrome (ARDS),meningitis, IgE-mediated diseases such as anaphylaxis and allergicrhinitis, encephalitis such as Rasmussen's encephalitis, uveitis,colitis such as microscopic colitis and collagenous colitis,glomerulonephritis (GN) such as membranous GN, idiopathic membranous GN,membranous proliferative GN (MPGN), including Type I and Type II, andrapidly progressive GN, allergic conditions, eczema, asthma, conditionsinvolving infiltration of T cells and chronic inflammatory responses,atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency,systemic lupus erythematosus (SLE) such as cutaneous SLE, lupus(including nephritis, cerebritis, pediatric, non-renal, discoid,alopecia), juvenile onset diabetes, multiple sclerosis (MS) such asspino-optical MS, allergic encephalomyelitis, immune responsesassociated with acute and delayed hypersensitivity mediated by cytokinesand T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis includingWegener's granulomatosis, agranulocytosis, vasculitis (including LargeVessel vasculitis (including Polymyalgia Rheumatica and Giant Cell(Takayasu's) Arteritis), Medium Vessel vasculitis (including Kawasaki'sDisease and Polyarteritis Nodosa), CNS vasculitis, and ANCA-associatedvasculitis, such as Churg-Strauss vasculitis or syndrome (CSS)),aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, immunehemolytic anemia including autoimmune hemolytic anemia (AIHA),pernicious anemia, pure red cell aplasia (PRCA), Factor VIII deficiency,hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseasesinvolving leukocyte diapedesis, CNS inflammatory disorders, multipleorgan injury syndrome, myasthenia gravis, antigen-antibody complexmediated diseases, anti-glomerular basement membrane disease,anti-phospholipid antibody syndrome, allergic neuritis, Bechet disease,Castleman's syndrome, Goodpasture's Syndrome, Lambert-Eaton MyasthenicSyndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnsonsyndrome, solid organ transplant rejection (including pretreatment forhigh panel reactive antibody titers, IgA deposit in tissues, andrejection arising from renal transplantation, liver transplantation,intestinal transplantation, cardiac transplantation, etc.), graft versushost disease (GVHD), pemphigoid bullous, pemphigus (including vulgaris,foliaceus, and pemphigus mucus-membrane pemphigoid), autoimmunepolyendocrinopathies, Reiter's disease, stiff-man syndrome, immunecomplex nephritis, IgM polyneuropathies or IgM mediated neuropathy,idiopathic thrombocytopenic purpura (ITP), thrombotic throbocytopenicpurpura (TTP), thrombocytopenia (as developed by myocardial infarctionpatients, for example), including autoimmune thrombocytopenia,autoimmune disease of the testis and ovary including autoimmune orchitisand oophoritis, primary hypothyroidism; autoimmune endocrine diseasesincluding autoimmune thyroiditis, chronic thyroiditis (Hashimoto'sThyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison'sdisease, Grave's disease, autoimmune polyglandular syndromes (orpolyglandular endocrinopathy syndromes), Type I diabetes also referredto as insulin-dependent diabetes mellitus (IDDM), including pediatricIDDM, and Sheehan's syndrome; autoimmune hepatitis, Lymphoidinterstitial pneumonitis (HIV), bronchiolitis obliterans(non-transplant) vs NSIP, Guillain-Barré Syndrome, Berger's Disease (IgAnephropathy), primary biliary cirrhosis, celiac sprue (glutenenteropathy), refractory sprue with co-segregate dermatitisherpetiformis, cryoglobulinemia, amylotrophic lateral sclerosis (ALS;Lou Gehrig's disease), coronary artery disease, autoimmune inner eardisease (AIED), autoimmune hearing loss, opsoclonus myoclonus syndrome(OMS), polychondritis such as refractory polychondritis, pulmonaryalveolar proteinosis, amyloidosis, giant cell hepatitis, scleritis,monoclonal gammopathy of uncertain/unknown significance (MGUS),peripheral neuropathy, paraneoplastic syndrome, channelopathies such asepilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness,periodic paralysis, and channelopathies of the CNS; autism, inflammatorymyopathy, and focal segmental glomerulosclerosis (FSGS).

The term “alkyl” refers to a straight chain or branched, saturated orunsaturated hydrocarbon having the indicated number of carbon atoms(e.g., “C₁-C₈ alkyl” refers to an alkyl group having from 1 to 8 carbonatoms). When the number of carbon atoms is not indicated, the alkylgroup has from 1 to 8 carbon atoms. Representative straight chain “C₁-C₈alkyl” groups include, but are not limited to, methyl, ethyl, n-propyl,n-butyl, n-pentyl, n-hexyl, n-heptyl and n-octyl; while branched C₁-C₈alkyls include, but are not limited to, -isopropyl, -sec-butyl,-isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl; unsaturated C₂-C₈alkyls include, but are not limited to, vinyl, -allyl, -1-butenyl,-2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl,-3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl,1-hexyl, 2-hexyl, 3-hexyl, -acetylenyl, -propynyl, -1-butynyl,-2-butynyl, -1-pentynyl, -2-pentynyl and -3-methyl-1 butynyl. An alkylgroup can be unsubstituted or substituted with one or more groupsincluding, but not limited to, —O—(C₁-C₈ alkyl), aryl, —C(O)R′,—OC(O)R′, —C(O)OR′, —C(O)NH₂, —OH—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′,—SO₃R′, —S(O)₂R′, —S(O)R′, —SR′, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂and —CN; where each R′ is independently selected from H, unsubstitutedC₁-C₈ alkyl and aryl.

“Alkenyl” refers to a C₂-C₁₈ hydrocarbon containing normal, secondary,tertiary or cyclic carbon atoms with at least one site of unsaturation,i.e. a carbon-carbon, sp² double bond. Examples include, but are notlimited to: ethylene or vinyl (—CH═CH₂), allyl (—CH₂CH═CH₂),cyclopentenyl (—C₅H₇), and 5-hexenyl (—CH₂CH₂CH₂CH₂CH═CH₂).

“Alkynyl” refers to a C₂-C₁₈ hydrocarbon containing normal, secondary,tertiary or cyclic carbon atoms with at least one site of unsaturation,i.e. a carbon-carbon, sp triple bond. Examples include, but are notlimited to: acetylenic (—C≡CH) and propargyl (—CH₂C≡CH).

“Alkylene” refers to a saturated, branched or straight chain or cyclichydrocarbon radical of 1-18 carbon atoms, and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkyleneradicals include, but are not limited to: methylene (—CH₂—), 1,2-ethyl(—CH₂CH₂—), 1,3-propyl (—CH₂CH₂CH₂—), 1,4-butyl (—CH₂CH₂CH₂CH₂—), andthe like. A “C₁-C₁₀ alkylene” is a straight chain, saturated hydrocarbongroup of the formula —(CH₂)₁₋₁₀—. Examples of a C₁-C₁₀ alkylene includemethylene, ethylene, propylene, butylene, pentylene, hexylene,heptylene, ocytylene, nonylene and decalene.

“Alkenylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkene. Typicalalkenylene radicals include, but are not limited to: 1,2-ethylene(—CH═CH—).

“Alkynylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkyne. Typicalalkynylene radicals include, but are not limited to: acetylene (—C≡C—),propargyl (—CH₂C≡C—), and 4-pentynyl (—CH₂CH₂CH₂C≡CH—).

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-20 carbonatoms derived by the removal of one hydrogen atom from a single carbonatom of a parent aromatic ring system. Some aryl groups are representedin the exemplary structures as “Ar”. Typical aryl groups include, butare not limited to, radicals derived from benzene, substituted benzene,naphthalene, anthracene, biphenyl, and the like. A carbocyclic aromaticgroup (aryl) or a heterocyclic aromatic group (heteroaryl) can beunsubstituted or substituted with one or more groups including, but notlimited to C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), —C(O)R′, —OC(O)R′, —C(O)OR′,—C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,-halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ isindependently selected from H, C₁-C₈ alkyl and unsubstituted aryl.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl, and thelike. The arylalkyl group comprises 6 to 20 carbon atoms, e.g., thealkyl moiety, including alkanyl, alkenyl or alkynyl groups, of thearylalkyl group is 1 to 6 carbon atoms and the aryl moiety is 6 to 14carbon atoms.

“Heteroarylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with a heteroaryl radical. Typicalheteroarylalkyl groups include, but are not limited to,2-benzimidazolylmethyl, 2-furylethyl, and the like. The heteroarylalkylgroup comprises 6 to 20 carbon atoms, e.g., the alkyl moiety, includingalkanyl, alkenyl or alkynyl groups, of the heteroarylalkyl group is 1 to6 carbon atoms and the heteroaryl moiety is 5 to 14 ring atoms,typically 1 to 3 heteroatoms selected from N, O, P, and S, with theremainder being carbon atoms. The heteroaryl moiety of theheteroarylalkyl group may be a monocycle having 3 to 7 ring members (2to 6 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S)or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S), for example: a bicyclo [4,5],[5,5], [5,6], or [6,6] system.

“Substituted alkyl”, “substituted aryl”, and “substituted arylalkyl”mean alkyl, aryl, and arylalkyl respectively, in which one or morehydrogen atoms are each independently replaced with a substituent.Typical substituents include, but are not limited to, —X, —R, —O⁻, —OR,—SR, —S⁻, —NR₂, —NR₃, ═NR, —CX₃, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO,—NO₂, ═N₂, —N₃, —NRC(═O)R, —C(═O)R, —C(═O)NR₂, —SO₃ ⁻, —SO₃H, —S(═O)₂R,—OS(═O)₂OR, —S(═O)₂NR, —S(═O)R, —OP(═O)(OR)₂, —P(═O)(OR)₂, —PO⁻ ₃,—PO₃H₂, —AsO₂H₂, —C(═O)R, —C(═O)X, —C(═S)R, —CO₂R, —CO₂ ⁻, —C(═S)OR,—C(═O)SR, —C(═S)SR, —C(═O)NR₂, —C(═S)NR₂, or —C(═NR)NR₂, where each X isindependently a halogen: F, Cl, Br, or I; and each R is independently H,C₁-C₂₀ alkyl, C₆-C₂₀ aryl, C₃-C₁₄ heterocycle, a protecting group or aprodrug moiety. Alkylene, alkenylene, and alkynylene groups as describedabove may also be similarly substituted.

“Heteroaryl” and “heterocycle” refer to a ring system in which one ormore ring atoms is a heteroatom, e.g., nitrogen, oxygen, and sulfur. Theheterocycle radical comprises 1 to 20 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S. A heterocycle may be amonocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected fromN, O, P, and S), for example: a bicyclo [4,5], [5,5], [5,6], or [6,6]system.

Heterocycles are described in Paquette, Leo A.; “Principles of ModernHeterocyclic Chemistry” (W. A. Benjamin, New York, 1968), particularlyChapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds,A series of Monographs” (John Wiley & Sons, New York, 1950 to present),in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc.(1960) 82:5566.

Examples of heterocycles include by way of example and not limitationpyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl,isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl,tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl,azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl,thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl,phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl.

By way of example and not limitation, carbon bonded heterocycles arebonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2,3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline. Still more typically, carbon bonded heterocycles include2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl,4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl,4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles arebonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine,2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline,3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline,piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of aisoindole, or isoindoline, position 4 of a morpholine, and position 9 ofa carbazole, or β-carboline. Still more typically, nitrogen bondedheterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl,1-pyrazolyl, and 1-piperidinyl.

A “C₃-C₈ heterocycle” refers to an aromatic or non-aromatic C₃-C₈carbocycle in which one to four of the ring carbon atoms areindependently replaced with a heteroatom from the group consisting of O,S and N. Representative examples of a C₃-C₈ heterocycle include, but arenot limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl,coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl,imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl,pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl andtetrazolyl. A C₃-C₈ heterocycle can be unsubstituted or substituted withup to seven groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen,—N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ is independentlyselected from H, —C₁-C₈ alkyl and aryl. “C₃-C₈ heterocyclo” refers to aC₃-C₈ heterocycle group defined above wherein one of the heterocyclegroup's hydrogen atoms is replaced with a bond. A C₃-C₈ heterocyclo canbe unsubstituted or substituted with up to six groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′,—OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

“Carbocycle” means a saturated or unsaturated ring having 3 to 7 carbonatoms as a monocycle or 7 to 12 carbon atoms as a bicycle. Monocycliccarbocycles have 3 to 6 ring atoms, still more typically 5 or 6 ringatoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g., arranged as abicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atomsarranged as a bicyclo [5,6] or [6,6] system. Examples of monocycliccarbocycles include cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl,and cyclooctyl. A “C₃-C₈ carbocycle” is a 3-, 4-, 5-, 6-, 7- or8-membered saturated or unsaturated non-aromatic carbocyclic ring.Representative C₃-C₈ carbocycles include, but are not limited to,-cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl,-cyclohexenyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl,-1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and-cyclooctadienyl. A C₃-C₈ carbocycle group can be unsubstituted orsubstituted with one or more groups including, but not limited to,—C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′,—C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,-halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl. A “C₃-C₈carbocyclo” refers to a C₃-C₈ carbocycle group defined above wherein oneof the carbocycle groups' hydrogen atoms is replaced with a bond.

An “arylene” is an aryl group which has two covalent bonds and can be inthe ortho, meta, or para configurations as shown in the followingstructures:

in which the phenyl group can be unsubstituted or substituted with up tofour groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂, —NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃, —NH₂,—NH(R′), —N(R′)₂ and —CN; wherein each R′ is independently selected fromH, —C₁-C₈ alkyl and aryl.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g., melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms, McGraw-HillBook Company, New York (1984); and Eliel and Wilen, Stereochemistry ofOrganic Compounds, John Wiley & Sons, Inc., New York (1994). Manyorganic compounds exist in optically active forms, i.e., they have theability to rotate the plane of plane-polarized light. In describing anoptically active compound, the prefixes D and L, or R and S, are used todenote the absolute configuration of the molecule about its chiralcenter(s). The prefixes d and l or (+) and (−) are employed to designatethe sign of rotation of plane-polarized light by the compound, with (−)or l meaning that the compound is levorotatory. A compound prefixed with(+) or d is dextrorotatory. For a given chemical structure, thesestereoisomers are identical except that they are mirror images of oneanother. A specific stereoisomer may also be referred to as anenantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

Examples of a “hydroxyl protecting group” include, but are not limitedto, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranylether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether,triethylsilyl ether, triisopropyl silyl ether, t-butyldimethyl silylether, triphenylmethyl silyl ether, acetate ester, substituted acetateesters, pivaloate, benzoate, methanesulfonate and p-toluenesulfonate.

“Leaving group” refers to a functional group that can be substituted byanother functional group. Such leaving groups are well known in the art,and examples include, but are not limited to, a halide (e.g., chloride,bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl),trifluoromethylsulfonyl (triflate), and trifluoromethylsulfonate.

Examples of a “patient” include, but are not limited to, a human, rat,mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird andfowl. In an exemplary embodiment, the patient is a human.

The phrase “pharmaceutically acceptable salt,” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound(e.g., a Drug, Drug-Linker compound, or a Drug-Linker-Ligand compound).The compound typically contains at least one amino group, andaccordingly acid addition salts can be formed with this amino group.Exemplary salts include, but are not limited to, sulfate, citrate,acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate,phosphate, acid phosphate, isonicotinate, lactate, salicylate, acidcitrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucuronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate(i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Apharmaceutically acceptable salt may involve the inclusion of anothermolecule such as an acetate ion, a succinate ion or other counterion.The counterion may be any organic or inorganic moiety that stabilizesthe charge on the parent compound. Furthermore, a pharmaceuticallyacceptable salt may have more than one charged atom in its structure.Instances where multiple charged atoms are part of the pharmaceuticallyacceptable salt can have multiple counter ions. Hence, apharmaceutically acceptable salt can have one or more charged atomsand/or one or more counterion.

“Pharmaceutically acceptable solvate” or “solvate” refer to anassociation of one or more solvent molecules and a compound of theinvention, e.g., an Exemplary Compound or Exemplary Conjugate. Examplesof solvents that form pharmaceutically acceptable solvates include, butare not limited to, water, isopropanol, ethanol, methanol, DMSO, ethylacetate, acetic acid, and ethanolamine.

The following abbreviations are used herein and have the indicateddefinitions: AE is auristatin E, Boc is N-(t-butoxycarbonyl), cit iscitrulline, dap is dolaproine, DCC is 1,3-dicyclohexylcarbodiimide, DCMis dichloromethane, DEA is diethylamine, DEAD isdiethylazodicarboxylate, DEPC is diethylphosphorylcyanidate, DIAD isdiisopropylazodicarboxylate, DIEA is N,N-diisopropylethylamine, dil isdolaisoleuine, DMAP is 4-dimethylaminopyridine, DME is ethyleneglycoldimethyl ether (or 1,2-dimethoxyethane), DMF is N,N-dimethylformamide,DMSO is dimethylsulfoxide, doe is dolaphenine, dov isN,N-dimethylvaline, DTNB is 5,5′-dithiobis(2-nitrobenzoic acid), DTPA isdiethylenetriaminepentaacetic acid, DTT is dithiothreitol, EDCI is1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EEDQ is2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ES-MS is electrospraymass spectrometry, EtOAc is ethyl acetate, Fmoc isN-(9-fluorenylmethoxycarbonyl), gly is glycine, HATU isO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate, HOBt is 1-hydroxybenzotriazole, HPLC is highpressure liquid chromatography, ile is isoleucine, lys is lysine,MeCN(CH₃CN) is acetonitrile, MeOH is methanol, Mtr is4-anisyldiphenylmethyl (or 4-methoxytrityl), nor is(1S,2R)-(+)-norephedrine, PAB is p-aminobenzyl, PBS isphosphate-buffered saline (pH 7.4), PEG is polyethylene glycol, Ph isphenyl, Pnp is p-nitrophenyl, MC is 6-maleimidocaproyl, phe isL-phenylalanine, PyBrop is bromo tris-pyrrolidino phosphoniumhexafluorophosphate, SEC is size-exclusion chromatography, Su issuccinimide, TBTU is O-benzotriazol-1-yl-N,N,N,N-tetramethyluroniumtetrafluoroborate, TFA is trifluoroacetic acid, TLC is thin layerchromatography, UV is ultraviolet, and val is valine.

The following linker abbreviations are used herein and have theindicated definitions: Val Cit or vc is a valine-citrulline, dipeptidesite in protease cleavable linker; PAB is p-aminobenzylcarbamoyl; (Me)vcis N-methyl-valine citrulline, where the linker peptide bond has beenmodified to prevent its cleavage by cathepsin B; MC(PEG)₆-OH ismaleimidocaproyl-polyethylene glycol; SPP is N-Succinimidyl4-(2-pyridylthio) pentanoate; and SMCC is N-Succinimidyl4-(N-maleimidomethyl)cyclohexane-1 carboxylate.

The terms “treat” or “treatment,” unless otherwise indicated by context,refer to both therapeutic treatment and prophylactic or preventativemeasures, wherein the object is to prevent or slow down (lessen) anundesired physiological change or disorder, such as the development orspread of cancer. For purposes of this invention, beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. Those in need of treatment include those alreadywith the condition or disorder as well as those prone to have thecondition or disorder or those in which the condition or disorder is tobe prevented.

In the context of cancer, the term “treating” includes any or all of:preventing growth of tumor cells, cancer cells, or of a tumor;preventing replication of tumor cells or cancer cells, lessening ofoverall tumor burden or decreasing the number of cancerous cells, andameliorating one or more symptoms associated with the disease.

In the context of an autoimmune disease, the term “treating” includesany or all of: preventing replication of cells associated with anautoimmune disease state including, but not limited to, cells thatproduce an autoimmune antibody, lessening the autoimmune-antibody burdenand ameliorating one or more symptoms of an autoimmune disease.

In the context of an infectious disease, the term “treating” includesany or all of: preventing the growth, multiplication or replication ofthe pathogen that causes the infectious disease and ameliorating one ormore symptoms of an infectious disease.

The following cytotoxic drug abbreviations are used herein and have theindicated definitions: “MMAF” isN-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine (MW 731.5);“MMAZ” is N-methylvaline-valine-dolaisoleuine-dolaproine with aphenylalanine analog at the C-terminus. Z is —NR⁹Z¹.

Embodiments of the Invention

Compounds and Conjugates

As noted in the Summary of the Invention, the present invention is drawnto a series of compounds and conjugates containing a drug compound (D).The drug compounds are useful as discrete entities, or can be conjugatedto Ligands (L, in some embodiments, antibodies), either directly orthrough a Linker Unit (LU). The Linker Unit can operate to provide asuitable release of D or spacing between D and L. Additionally, someLinker Units can have multiple attached drugs (e.g., one to fourattached drugs can be represented as -LU-(D)₁₋₄).

In one group of embodiments, the invention provides compounds havingFormula I:L-(D)_(p)  (I)or a pharmaceutically acceptable salt or solvate thereof, wherein L- isa Ligand unit; p is an integer of from 1 to about 20; and D is a drugmoiety having Formula D:

wherein: R² is selected from the group consisting of H and C₁-C₈ alkyl;R³ is selected from the group consisting of H, C₁-C₈ alkyl, C₃-C₈carbocycle, aryl, X¹-aryl, X¹—(C₃-C₈ carbocycle), C₃-C₈ heterocycle andX¹—(C₃-C₈ heterocycle); R⁴ is selected from the group consisting of H,C₁-C₉ alkyl, C₃-C₈ carbocycle, aryl, X¹-aryl, X¹—(C₃-C₈ carbocycle),C₃-C₈ heterocycle and X¹—(C₃-C₈ heterocycle); R⁵ is selected from thegroup consisting of H and methyl; or R⁴ and R⁵ jointly form acarbocyclic ring and have the formula —(CR^(a)R^(b))_(n)— wherein R^(a)and R^(b) are independently selected from the group consisting of H,C₁-C₈ alkyl and C₃-C₈ carbocycle and n is selected from the groupconsisting of 2, 3, 4, 5 and 6; R⁶ is selected from the group consistingof H and C₁-C₈ alkyl; R⁷ is selected from the group consisting of H,C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, X¹-aryl, X¹—(C₃-C₈ carbocycle),C₃-C₈ heterocycle and X¹—(C₃-C₈ heterocycle); each R⁸ is independentlyselected from the group consisting of H, OH, C₁-C₈ alkyl, C₃-C₈carbocycle and O—(C₁-C₈ alkyl); each X¹ is independently C₁-C₁₀alkylene; and the moiety —NR⁹Z¹ is a phenylalanine bioisostere with amodified amino acid side chain.

In one embodiment, the phenylalanine bioisostere moiety

is selected from the group consisting of:

wherein

represents a single or double bond;

R⁹ is selected from the group consisting of H and an amino protectinggroup;

R¹⁰ is selected from the group consisting of H and —(CR¹³R¹⁴)_(x)R¹⁵;

each R¹¹ is independently selected from the group consisting of H,C₁-C₂₀alkyl, halogen, aryl, arylC₁-C₂₀alkyl, C₁-C₁₀haloalkyl, OR¹⁶ andN(R¹⁶)₂;

R¹² is selected from the group consisting of H, C₁-C₂₀alkyl, halogen,aryl, arylC₁-C₂₀alkyl, arylC₂-C₂₀alkenyl, arylC₂-C₂₀alkynyl, OR¹⁶,N(R¹⁶)₂ and —C(O)R¹⁶;

each R¹³ and R¹⁴ is independently selected from the group consisting ofH, C₁-C₂₀ alkyl, halogen, arylalkyl, C₁-C₁₀haloalkyl, OR¹⁶, SR¹⁶,N(R¹⁶)₂, —OC(O)R¹⁶, —N(R¹⁶)C(O)R¹⁶, —COOR¹⁶, —CON(R¹⁶)₂, X¹—SO₃H,X¹—SO₃—C₁-C₂₀alkyl, X¹—OSO₃H, X¹—OSO₃—C₁-C₂₀alkyl, X¹—SO₂—C₁-C₂₀alkyl,X¹—SO—C₁-C₂₀ alkyl, —OP(O)(OR¹⁶)₂, —OP(O)(NR¹⁶)₂, —OP(O)N(R¹⁶)₂OR¹⁶,—OP(O)(R¹⁶)OR¹⁶, —OP(O)(R¹⁶)N(R¹⁶)₂, —P(O)(OR¹⁶)₂, —P(O)(NR¹⁶)₂,—P(O)N(R¹⁶)₂OR¹⁶, C₁-C₂₀ alkyl, C₃-C₈carbocycle, aryl, X¹-aryl,X¹—C₃-C₈carbocycle, C₃-C₂₀heterocycle and X¹—C₃-C₈heterocycle;

or R¹³ and R¹⁴ are combined together to form a member selected from thegroup consisting of ═O, ═N—NH—R¹⁷, ═N—NH—C(O)—R¹⁷ and a C₃-C₈carbocycle;

each R¹⁵ is independently selected from the group consisting of H,C₁-C₂₀alkyl, C₃-C₈ carbocycle, aryl, X¹-aryl, C₁-C₂₀ alkyl-C₃-C₈carbocycle, C₃-C₂₀ heterocycle, X¹—C₃-C₈ heterocycle, —COOR¹⁶,—CON(R¹⁶)₂, —C(O)R¹⁶ and Y¹(CR¹³R¹⁴)_(x)R¹⁸; and the carbocycle, aryland heterocycle portions are optionally substituted with from one tothree R¹² groups.

each R¹⁶ is independently H or C₁-C₂₀alkyl;

R¹⁷ is selected from the group consisting of H, C₁-C₂₀alkyl,C₃-C₈carbocycle, aryl, X¹-aryl, C₁-C₂₀alkyl-C₃-C₈carbocycle,C₃-C₈heterocycle and X¹—C₃-C₈heterocycle;

each R¹⁸ is independently selected from the group consisting of H,C₁-C₂₀alkyl, C₃-C₈carbocycle, aryl, X¹-aryl, X¹—C₃-C₈carbocycle,C₃-C₂₀heterocycle, X¹—C₃-C₈heterocycle, —COOR¹⁶, —CON(R¹⁶)₂ and—C(O)R¹⁶;

Y¹ is O, S, NR¹⁶, SO, SO₂ or Se;

each X¹ is independently C₁-C₁₀ alkylene;

the subscript x is an integer from 0 to 10;

the subscripts n, o, q, r, s, t and u are integers independently from 0to 2;

Z² is COZ³R¹⁹;

Z³ is O, S, NH, or NR²⁰, wherein R²⁰ is C₁-C₈ alkyl;

R¹⁹ is selected from H, C₁-C₂₀ alkyl, aryl, C₃-C₈ heterocycle,—(X¹O)_(v)—R²², or —(X¹O)_(v)—CH(R²³)₂;

v is an integer ranging from 1-1000;

R²² is H or C₁-C₈ alkyl;

each R²³ is independently H, COOH, —(CH₂)_(l)—N(R²⁴)₂, —(CH₂)_(l)—SO₃Hor —(CH₂)_(l)—SO₃—C₁-C₈ alkyl; and

each R²⁴ is independently H, C₁-C₈ alkyl or —(CH₂)_(l)—COOH; where; l isan integer ranging from 0 to 6; with the proviso when n and o are 0, andR¹¹ is H, then R¹⁰ is other than CH₂-aryl or CH₂—C₃-C₈heterocycle.

In one embodiment, R⁹ is selected from the group consisting of H, C₁-C₂₀alkyl, C₃-C₈ carbocyclyl, X¹-aryl, X¹—(C₃-C₈-carbocyclyl) andX¹−C₃-C₈-heterocyclyl. In another embodiment R⁹ is H.

In one embodiment, the phenylalanine bioisostere moiety is

wherein R⁹ is H; R¹⁰ is benzyl; R¹¹ is H; Z² is CO₂H; the subscript n isan integer of from 0 to 2; and the subscript o is an integer of from 0to 1 with the proviso that n+o is at least 1.

In one embodiment, the phenylalanine bioisostere moiety is selected fromthe group consisting of

In one embodiment, the phenylalanine bioisostere moiety is

wherein R⁹ is H or an amino protecting group; R¹⁰ is H and—(CR¹³R¹⁴)_(x)R¹⁵; each R¹¹ is independently H, C₁-C₂₀alkyl, halogen,aryl, arylC₁-C₂₀alkyl, C₁-C₁₀haloalkyl, OR¹⁶ and N(R¹⁶)₂; R¹² is H,C₁-C₂₀alkyl, halogen, aryl, arylC₁-C₂₀alkyl, arylC₂-C₂₀alkenyl,arylC₂-C₂₀alkynyl, OR¹⁶, N(R¹⁶)₂ and —C(O)R¹⁶; each R¹³ and R¹⁴ isindependently H, C₁-C₂₀ alkyl, halogen, arylalkyl, C₁-C₁₀haloalkyl,OR¹⁶, SR¹⁶, N(R¹⁶)₂, —OC(O)R¹⁶, —N(R¹⁶)C(O)R¹⁶, —COOR¹⁶, —CON(R¹⁶)₂,—X¹—SO₃H, —X¹—SO₃—C₁-C₂₀alkyl, —X¹—OSO₃H, —X¹—OSO₃—C₁-C₂₀alkyl,—X¹—SO₂—C₁-C₂₀alkyl, —X¹—SO—C₁-C₂₀ alkyl, —OP(O)(OR¹⁶)₂, —OP(O)(NR¹⁶)₂,—OP(O)N(R¹⁶)₂OR¹⁶, —OP(O)(R¹⁶)OR¹⁶, —OP(O)(R¹⁶)N(R¹⁶)₂, —P(O)(OR¹⁶)₂,—P(O)(NR¹⁶)₂, —P(O)N(R¹⁶)₂OR¹⁶, C₁-C₂₀ alkyl, C₃-C₈carbocycle, aryl,—X¹-aryl, —X¹—C₃-C₈carbocycle, C₃-C₈ heterocycle and —X¹—C₃-C₈heterocycle; or R¹³ and R¹⁴ are combined together to form a memberselected from the group consisting of ═O, ═N—NH—R¹⁷, ═N—NH—C(O)—R¹⁷ anda C₃-C₈ carbocycle; each R¹⁵ is independently H, C₁-C₂₀alkyl, C₃-C₈carbocycle, aryl, —X¹-aryl, C₁-C₂₀ alkyl-C₃-C₈ carbocycle, C₃-C₈heterocycle, —X¹—C₃-C₈ heterocycle, —COOR¹⁶, —CON(R¹⁶)₂, —C(O)R¹⁶ and—Y¹(CR¹³R¹⁴)_(x)R¹⁸ wherein the carbocycle, aryl and heterocycleportions are optionally substituted with from one to three R¹² groups;each R¹⁶ is independently H or C₁-C₂₀alkyl; R¹⁷ is H, C₁-C₂₀alkyl,C₃-C₈carbocycle, aryl, X¹-aryl, C₁-C₂₀alkyl-C₃-C₈carbocycle,C₃-C₈heterocycle and X¹—C₃-C₈heterocycle; each R¹⁸ is independently H,C₁-C₂₀alkyl, C₃-C₈carbocycle, aryl, X¹-aryl, X¹—C₃-C₈carbocycle, C₃-C₈heterocycle, X¹—C₃—C heterocycle, —COOR¹⁶, —CON(R¹⁶)₂ and —C(O)R¹⁶; Y¹is O, S, NR¹⁶, SO, SO₂ or Se; each X¹ is independently C₁-C₁₀ alkylene;the subscript x is an integer from 0 to 10; the subscripts n, o, q, r,s, t and u are integers independently from 0 to 2 with the proviso thatn+o is at least 1; Z² is COZ³R¹⁹; Z³ is O, S, NH, or NR²⁰, wherein R²⁰is C₁-C₈ alkyl; R¹⁹ is selected from H, C₁-C₂₀ alkyl, aryl, C₃-C₈heterocycle, —(X¹O)_(v)—R²², or —(X¹O)_(v)—CH(R²³)₂; v is an integerranging from 1-1000; R²² is H or C₁-C₈ alkyl; and each R²³ isindependently H, COOH, —(CH₂)_(l)—N(R⁴)₂, —(CH₂)_(l)—SO₃H or—(CH₂)_(l)—SO₃—C₁-C₈ alkyl; each R²⁴ is independently H, C₁-C₈ alkyl or—(CH₂)_(l)—COOH; where; l is an integer ranging from 0 to 6; with theproviso when n and o are 0, R¹¹ is H the R¹⁰ is other than CH₂-aryl orCH₂—C₃-C₈heterocycle.

In one embodiment, the phenylalanine bioisostere moiety is

wherein R¹² and R¹⁴ are as described above; Z² is CO₂H; and thesubscript x is an integer of from 0 to 2.

In one embodiment, the phenylalanine bioisostere moiety is

wherein R¹⁰ is CH₂—C₃-C₈heterocycle or CH₂-aryl; R¹¹ is H; and Z² is asdescribed above. In some embodiments, R¹⁰ is selected from the groupconsisting of:

wherein R¹² is as described above; each X² is independently selectedfrom the group consisting of N, NR¹⁶, S, O, CR¹⁶ and CHR¹⁶; and thesubscript z is an integer of from 0 to 2. In other embodiments, R¹⁰ isselected from the group consisting of:

wherein R¹² is as described above; each X² is independently selectedfrom the group consisting of N, NR¹⁶, S, O, CR¹⁶ and CHR¹⁶; and thesubscript z is an integer of from 0 to 2; and no more than two adjacentX² groups are other than CR¹⁶ or CHR¹⁶. In still other embodiments, R¹⁰is selected from the group consisting of:

wherein R¹² is selected from the group: H, alkyl, halogen, amino,carboxy, amido, carboethoxy, formyl, phenyl, E-2-phenylethenyl,Z-2-phenylethenyl, and 2-phenylethynyl.

In one group of embodiments, the phenylalanine bioisostere moiety isselected from the group consisting of:

In one embodiment, the phenylalanine bioisostere moiety is selected fromthe group consisting of:

In one embodiment, the phenylalanine bioisostere moiety is the amide ofan α-amino acid selected from the group consisting of:

In one embodiment, the phenylalanine bioisostere moiety is the α-aminoamide of an amino acid selected from the group consisting of:4-chloro-phenylalanine, 4-fluoro-phenylalanine, 4-nitro-phenylalanine,N-α-methyl-phenylalanine, α-methyl-phenylalanine, glutamic acid,aspartic acid, tryptophan, isoleucine, leucine, methionine, tyrosine,glutamine, threonine, valine, asparagine, phenylglycine,O-benzyl-serine, O-t-butyl-serine, O-t-butyl-threonine,homophenylalanine, methionine-DL-sulfoxide, methionine-sulfone,α-aminobutyric acid, α-aminoisobutyric acid,4-amino-1-piperidine-4-carboxylic acid,4-amino-tetrahydropyran-4-carboxylic acid, aspartic acid,benzothiazol-2-yl-alanine, α-t-butyl-glycine, cyclohexylalanine,norleucine, norvaline, S-acetamidomethyl-penicillamine,β-3-piperidin-3-yl-alanine, piperidinyl-glycine, pyrrolidinyl-alanine,selenocysteine, tetrahydropyran-4-yl-glycine, O-benzyl-threonine,O-t-butyl-tyrosine, 3-(p-acetylphenyl)alanine, 3-phenylserine, and1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid.

In one embodiment, the phenylalanine bioisostere moiety is

wherein Z² is CO₂H; R¹⁰ is benzyl; and the subscripts q, r and sindependently are integers of from 0 to 1.

In one embodiment, the phenylalanine bioisostere moiety is selected fromthe group consisting of:

In one embodiment, the phenylalanine bioisostere moiety is

wherein Z² is CO₂H; R¹⁰ is benzyl; R¹¹ is H; and the subscripts t and uindependently are integers of from 1 to 3.

In one embodiment, the phenylalanine bioisostere moiety is selected fromthe group consisting of

In one embodiment, the phenylalanine bioisostere moiety is

wherein Z² is CO₂H; R¹² is as described above; and the subscripts q, rand s independently are integers of from 1 to 3.

In one embodiment, the phenylalanine bioisostere moiety is:

In one embodiment, the phenylalanine bioisostere moiety is

wherein Z² is CO₂H; R¹⁰ is benzyl; and R¹² is as described above.

In one embodiment, the phenylalanine bioisostere moiety is

In one embodiment, the phenylalanine bioisostere moiety is

wherein Z² is CO₂H; R¹² is as described above; and the subscripts q, rand s independently are integers of from 1 to 3.

In one embodiment, the phenylalanine bioisostere moiety is

In a related aspect, the present invention provides conjugates in whichthe compounds further comprise a Linker unit (LU), the conjugates havingthe formula:L-(LU-(D)₁₋₄)_(p)or a pharmaceutically acceptable salt or solvate thereof wherein L is aLigand unit; -LU- is a Linker unit; and D is a Drug unit, as set forthherein.

In another related aspect, the present invention provides conjugateshaving the formula:LU-(D)₁₋₄or a pharmaceutically acceptable salt or solvate thereof wherein, -LU-is a Linker unit; and D is a drug moiety having the Formula D:

according to any of the above embodiments.

In one embodiment, Drug-Linker-Ligand Conjugates are provided that haveFormula Ia:L

A_(a)-W_(w)—Y_(y)-D)_(p)  Iaor a pharmaceutically acceptable salt or solvate thereof, wherein L- isa Ligand unit; -A_(a)-W_(w)—Y_(y)— is a Linker unit (LU), wherein -A- isa Stretcher unit, the subscript a is 0 or 1, each —W— is independentlyan Amino Acid unit, w is an integer ranging from 0 to 12, —Y— is aSpacer unit, and y is 0, 1 or 2; p is an integer of from 1 to about 20;and D is a Drug unit having the Formula D:

wherein R² is selected from the group consisting of H and C₁-C₈ alkyl;R³ is selected from the group consisting of H, C₁-C₈ alkyl, C₃-C₈carbocycle, aryl, C₁-C₈ alkyl-aryl, X¹—(C₃-C₈ carbocycle), C₃-C₈heterocycle and X¹—(C₃-C₈ heterocycle); R⁴ is selected from the groupconsisting of H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, X¹-aryl, C₁-C₈alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and X¹—(C₃-C₈ heterocycle);R⁵ is selected from the group consisting of H and methyl; or R⁴ and R⁵jointly form a carbocyclic ring and have the formula—(CR^(a)R^(b))_(n)—, wherein R^(a) and R^(b) are independently selectedfrom the group consisting of H, C₁-C₈ alkyl and C₃-C₈ carbocycle and nis selected from the group consisting of 2, 3, 4, 5 and 6; R⁶ isselected from the group consisting of H and C₁-C₈ alkyl; R⁷ is selectedfrom the group consisting of H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,X¹-aryl, X¹—(C₃-C₈ carbocycle), C₃-C₈ heterocycle and X¹—(C₃-C₈heterocycle); each R³ is independently selected from the groupconsisting of H, OH, C₁-C₈ alkyl, C₃-C₈ carbocycle and O—(C₁-C₈ alkyl);each X¹ is independently C₁-C₁₀ alkylene; and the moiety —NR⁹Z¹ is aphenylalanine bioisostere of any of the above embodiments.

Another aspect of the invention are the Drug Compounds having theFormula Ib. These drug compounds are those described above wherein thewavy line is replaced by a hydrogen atom. Specifically, the compoundsare represented below:

or pharmaceutically acceptable salts or solvates thereof, wherein, R² isselected from H and C₁-C₈ alkyl; R³ is selected from H, C₁-C₈ alkyl,C₃-C₈ carbocycle, aryl, C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈carbocycle), C₃-C₈ heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle); R⁴is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle); R⁵ is selected from H and methyl; or R⁴ andR⁵ jointly form a carbocyclic ring and have the formula—(CR^(a)R^(b))_(n)—, wherein R^(a) and R^(b) are independently selectedfrom H, C₁-C₈ alkyl and C₃-C₈ carbocycle and n is selected from 2, 3, 4,5 and 6; R⁶ is selected from H and C₁-C₈ alkyl; R⁷ is selected from H,C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈ alkyl-aryl, C₁-C₈alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈ alkyl-(C₃-C₈heterocycle); each R⁸ is independently selected from the groupconsisting of H, OH, C₁-C₈ alkyl, C₃-C₈ carbocycle and O—(C₁-C₈ alkyl);and the moiety —NR⁹Z¹ is a phenylalanine bioisostere of any of the aboveembodiments.

In one embodiment, R³, R⁴ and R⁷ are independently isopropyl orsec-butyl and R⁵ is —H. In an exemplary embodiment, R³ and R⁴ are eachisopropyl, R⁵ is —H, and R⁷ is sec-butyl.

In another embodiment, R² and R⁶ are each methyl, and R⁹ is —H.

In still another embodiment, each occurrence of R⁸ is —OCH₃.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R² and R⁶ areeach methyl, R⁵ is —H, R⁷ is sec-butyl, each occurrence of R⁸ is —OCH₃,and R⁹ is —H.

Illustrative Compounds of Formula (Ib), each of which may be used asdrug moieties (D) in an ADC, include compounds having the followingstructures:

and pharmaceutically acceptable salts or solvates thereof.

In yet another aspect, Drug-Linker-Ligand Conjugates are provided inwhich the Ligand is an antibody. In this aspect, the conjugates arerepresented by Formula Ia′:Ab

A_(a)-W_(w)—Y_(y)-D)_(p)  Formula Ia′or pharmaceutically acceptable salts or solvates thereof, wherein Ab isan antibody, A is a Stretcher unit, a is 0 or 1, each W is independentlyan Amino Acid unit, w is an integer ranging from 0 to 12, Y is a Spacerunit, and y is 0, 1 or 2, p is an integer of from 1 to about 20, and Dis a Drug moiety of Formula D:

wherein R², R³, R⁴, R⁵, R⁶, R⁷, each R⁸, and —N(R⁹)Z¹ have the meaningsprovided above.

The antibody Ab can be any antibody covalently attached to one or moredrug units. For example, Ab can be an antibody that specifically bindsto CD20, CD30, CD33, CD40, CD70, BCMA, or Lewis Y antigen.

In one embodiment —W_(w)— is -Val-Cit-.

In another embodiment, R³, R⁴ and R⁷ are independently isopropyl orsec-butyl and R⁵ is —H. In an exemplary embodiment, R³ and R⁴ are eachisopropyl, R⁵ is —H, and R⁷ is sec-butyl. In yet another embodiment, R²and R⁶ are each methyl, and R⁹ is —H.

In still another embodiment, each occurrence of R⁸ is —OCH₃.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R² and R⁶ areeach methyl, R⁵ is —H, R⁷ is sec-butyl, each occurrence of R⁸ is —OCH₃,and R⁹ is —H.

In one aspect, the antibody Ab is chimeric AC10, chimeric BR96, chimericS2C6, chimeric 1F6, chimeric 2F2, humanized AC10, humanized BR96,humanized S2C6, humanized 1F6, M195, humanized M195 or humanized 2F2.

Exemplary embodiments of Formula Ia′ have the following structures:

The drug loading is represented by p, the average number of drugmolecules per ligand (e.g., an antibody) (e.g., of Formula I, Ia, Ia′).Drug loading may range from 1 to 20 Drug units (D) per Ligand (e.g., Abor mAb). The Drug unit may be conjugated directly or indirectly to theLigand unit (e.g., via a Linker unit). Compositions of Formula Ia andFormula Ia′ include collections of antibodies conjugated with a range ofdrugs, from 1 to 20.

In some embodiments, p is from about 1 to about 8 Drug units per Ligandunit. In some embodiments, p is from about 2 to about 8 Drug units perLigand unit. In some embodiments, p is from about 2 to about 6, or 2 toabout 4 Drug units per Ligand unit. In some embodiments, p is about 2,about 4, about 6 or about 8 Drug units per Ligand unit

The average number of Drugs units per Ligand unit in preparation ofconjugation reactions may be characterized by conventional means such asmass spectroscopy, ELISA assay, and HPLC. The quantitative distributionof Ligand-Drug-Conjugates in terms of p may also be determined. In someinstances, separation, purification, and characterization of homogeneousLigand-Drug-conjugates where p is a certain value fromLigand-Drug-Conjugates with other drug loadings may be achieved by meanssuch as reverse phase HPLC or electrophoresis.

Returning to Formula Ia′, the conjugates comprising an antibodycovalently attached to one or more Drug units (moieites): A, a, W, w, Yand y are as described above. The antibody drug conjugate compoundsinclude pharmaceutically acceptable salts or solvates thereof.

The drug loading is represented by p, the average number of Drugs unitsper antibody in a molecule of Formula I. Drug loading may range from 1to 20 drugs (D) per antibody (Ab or mAb). The Drug Unit may beconjugated directly or indirectly to the Ligand unit (e.g., via a Linkerunit). Compositions of ADC of Formula Ic include collections ofantibodies conjugated with a range of drugs, from 1 to 20. In someembodiments, p is from about 1 to about 8 Drug units per antibody. Insome embodiments, p is from about 2 to about 8 Drug units per antibody.In some embodiments, p is from about 2 to about 6, or 2 to about 4 Drugunits per antibody. In some embodiments, p is about 2, about 4, about 6or about 8 Drug units per antibody.

The average number of drugs per antibody in preparations of ADC fromconjugation reactions may be characterized by conventional means such asUV/visible spectroscopy, mass spectrometry, ELISA assay, and HPLC. Thequantitative distribution of ADC in terms of p may also be determined.In some instances, separation, purification, and characterization ofhomogeneous ADC where p is a certain value from ADC with other drugloadings may be achieved by means such as reverse phase HPLC orelectrophoresis.

For some antibody drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in the exemplary embodiments above, an antibody mayhave only one or several cysteine thiol groups, or may have only one orseveral sufficiently reactive thiol groups through which a linker may beattached.

Typically, fewer than the theoretical maximum of drug moieties areconjugated to an antibody during a conjugation reaction. An antibody maycontain, for example, many lysine residues that do not react with thedrug-linker intermediate or linker reagent. Only the most reactivelysine groups may react with an amine-reactive linker reagent.Generally, antibodies do not contain many, if any, free and reactivecysteine thiol groups which may be linked to a drug moiety. Mostcysteine thiol residues in the antibodies exist as disulfide bridges andmust be reduced with a reducing agent such as dithiothreitol (DTT).Additionally, the antibody must be subjected to denaturing conditions toreveal reactive nucleophilic groups such as lysine or cysteine. Theloading (drug/antibody ratio) of an ADC may be controlled in severaldifferent manners, including: (i) limiting the molar excess ofdrug-linker intermediate or linker reagent relative to antibody, (ii)limiting the conjugation reaction time or temperature, and (iii) partialor limiting reductive conditions for cysteine thiol modification.

Where more than one nucleophilic group reacts with a drug-linkerintermediate, or linker reagent followed by drug moiety reagent, thenthe resulting product is a mixture of ADC compounds with a distributionof one or more drug moieties attached to an antibody. The average numberof drugs per antibody may be calculated from the mixture by dual ELISAantibody assay, specific for antibody and specific for the drug.Individual ADC molecules may be identified in the mixture by massspectroscopy, and separated by HPLC, e.g., hydrophobic interactionchromatography (“Effect of drug loading on the pharmacology,pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate”,Hamblett, K. J., et al, Abstract No. 624, American Association forCancer Research; Hamblett et al., 2004, Cancer Research 10:7063; 2004Annual Meeting, Mar. 27-31, 2004, Proceedings of the AACR, Volume 45,March 2004; “Controlling the Location of Drug Attachment inAntibody-Drug Conjugates”, Alley, S. C., et al, Abstract No. 627,American Association for Cancer Research; 2004 Annual Meeting, Mar.27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). Thus, ahomogeneous ADC with a single loading value may be isolated from theconjugation mixture by electrophoresis or chromatography.

The Linker Unit (LU)

A “Linker unit” (LU) is a bifunctional compound which can be used tolink a Drug unit and a Ligand unit to form Drug-Linker-LigandConjugates, or which are useful in the formation of immunoconjugatesdirected against tumor associated antigens. Such immunoconjugates allowthe selective delivery of toxic drugs to tumor cells.

In one embodiment, the Linker unit of the Drug-Linker Compound andDrug-Linker-Ligand Conjugate has the formula:-A_(a)-W_(w)—Y_(y)—wherein -A- is a Stretcher unit; a is 0 or 1; each —W— is independentlyan Amino Acid unit; w is independently an integer ranging from 0 to 12;—Y— is a Spacer unit; and y is 0, 1 or 2.

In the Drug-Linker-Ligand Conjugate, the Linker is serves to attach theDrug moiety and the Ligand unit.

The Stretcher Unit

The Stretcher unit (-A-), when present, is capable of linking a Ligandunit to an amino acid unit (—W—). In this regard a Ligand (L) has afunctional group that can form a bond with a functional group of aStretcher. Useful functional groups that can be present on a ligand,either naturally or via chemical manipulation include, but are notlimited to, sulfhydryl (—SH), amino, hydroxyl, carboxy, the anomerichydroxyl group of a carbohydrate, and carboxyl. In one aspect, theLigand functional groups are sulfhydryl and amino. Sulfhydryl groups canbe generated by reduction of an intramolecular disulfide bond of aLigand. Alternatively, sulfhydryl groups can be generated by reaction ofan amino group of a lysine moiety of a Ligand using 2-iminothiolane(Traut's reagent) or another sulfhydryl generating reagent.

In one embodiment, the Stretcher unit forms a bond with a sulfur atom ofthe Ligand unit. The sulfur atom can be derived from a sulfhydryl groupof a Ligand. Representative Stretcher units of this embodiment aredepicted within the square brackets of Formulas IIIa and IIIb, whereinL-, —W—, —Y—, -D, w and y are as defined above, and R¹⁷ is selected from—C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-, —O—(C₁-C₈ alkyl)-, -arylene-,—C₁-C₁₀ alkylene-arylene-, -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀alkylene-(C₃-C₈ carbocyclo)-, —(C₃-C₈ carbocyclo)-C₁-C₁₀ alkylene-,—C₃-C₈ heterocyclo-, —C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈heterocyclo)-C₁-C₁₀ alkylene-, —(CH₂CH₂O)_(r)—, and —(CH₂CH₂O)_(r)—CH₂—;and r is an integer ranging from 1-10. It is to be understood from allthe exemplary embodiments of Formula Ia, such as III-VI, that even wherenot denoted expressly, from 1 to 20 drug moieties are linked to a Ligand(p=1-20).

L

CH₂—CONH—R¹⁷—C(O)—W_(w)—Y_(y)-D]_(p)  IIIb

An illustrative Stretcher unit is that of Formula IIIa wherein R¹⁷ is—(CH₂)₅—:

Another illustrative Stretcher unit is that of Formula IIIa wherein R¹⁷is —(CH₂CH₂O)_(r)—CH₂—; and r is 2:

Still another illustrative Stretcher unit is that of Formula IIIbwherein R¹⁷ is —(CH₂)₅—:

In another embodiment, the Stretcher unit is linked to the Ligand unitvia a disulfide bond between a sulfur atom of the Ligand unit and asulfur atom of the Stretcher unit. A representative Stretcher unit ofthis embodiment is depicted within the square brackets of Formula IV,wherein R¹⁷, L-, —W—, —Y—, -D, w and y are as defined above.L

S—R¹⁷—C(O)—W_(w)—Y_(y)-D]_(p)  IV

In yet another embodiment, the reactive group of the Stretcher containsa reactive site that can form a bond with a primary or secondary aminogroup of a Ligand. Example of these reactive sites include, but are notlimited to, activated esters such as succinimide esters, 4-nitrophenylesters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides,acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates.Representative Stretcher units of this embodiment are depicted withinthe square brackets of Formulas Va-Vc, wherein —R¹⁷—, L-, —W—, —Y—, -D,w and y are as defined above;L

C(O)NH—R¹⁷—C(O)—W_(w)—Y_(y)-D]_(p)  VaL

C(S)NH—R¹⁷—C(O)—W_(w)—Y_(y)-D]_(p)  VbL

C(O)—R¹⁷—C(O)—W_(w)—Y_(y)-D]_(p)  Vc

In yet another aspect, the reactive group of the Stretcher contains areactive site that is reactive to a modified carbohydrate's (—CHO) groupthat can be present on a Ligand. For example, a carbohydrate can bemildly oxidized using a reagent such as sodium periodate and theresulting (—CHO) unit of the oxidized carbohydrate can be condensed witha Stretcher that contains a functionality such as a hydrazide, an oxime,a primary or secondary amine, a hydrazine, a thiosemicarbazone, ahydrazine carboxylate, and an arylhydrazide such as those described byKaneko, T. et al. (1991) Bioconjugate Chem 2:133-41. RepresentativeStretcher units of this embodiment are depicted within the squarebrackets of Formulas VIa, VIb, and VIc, wherein —R¹⁷—, L-, —W—, —Y—, -D,w and y are as defined above.

The Amino Acid Unit

The Amino Acid unit (—W—), when present, links the Stretcher unit to theSpacer unit if the Spacer unit is present, links the Stretcher unit tothe Drug moiety if the Spacer unit is absent, and links the Ligand unitto the Drug unit if the Stretcher unit and Spacer unit are absent.

W_(w)— is a dipeptide, tripeptide, tetrapeptide, pentapeptide,hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide,undecapeptide or dodecapeptide unit. Each —W— unit independently has theformula denoted below in the square brackets, and w is an integerranging from 0 to 12:

wherein R¹⁹⁰ is hydrogen, methyl, isopropyl, isobutyl, sec-butyl,benzyl, p-hydroxybenzyl, —CH₂OH, —CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂,—CH₂COOH, —CH₂CH₂CONH₂, —CH₂CH₂COOH, —(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂,—(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO, —(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂,—(CH₂)₄NHCOCH₃, —(CH₂)NHCHO, —(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂,—CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3-pyridylmethyl-,4-pyridylmethyl-, phenyl, cyclohexyl,

The Amino Acid unit can be enzymatically cleaved by one or more enzymes,including a tumor-associated protease, to liberate the Drug unit (-D),which in one embodiment is protonated in vivo upon release to provide aDrug (D).

Illustrative W_(w) units are represented by formulas (VII)-(IX):

wherein R²⁰⁰ and R²⁰¹ are as follows:

(VIII)

R²⁰⁰ R²⁰¹ benzyl (CH₂)₄NH₂; methyl (CH₂)₄NH₂; isopropyl (CH₂)₄NH₂;isopropyl (CH₂)₃NHCONH₂; benzyl (CH₂)₃NHCONH₂; isobutyl (CH₂)₃NHCONH₂;sec-butyl (CH₂)₃NHCONH₂;

(CH₂)₃NHCONH₂; benzyl methyl; and benzyl (CH₂)₃NHC(═NH)NH₂;wherein R²⁰⁰, R²⁰¹ and R²⁰² are as follows:

(IX)

R²⁰⁰ R²⁰¹ R²⁰² benzyl benzyl (CH₂)₄NH₂; isopropyl benzyl (CH₂)₄NH₂; andH benzyl (CH₂)₄NH₂;wherein R²⁰⁰, R²⁰¹, R²⁰² and R²⁰³ are as follows:

R²⁰⁰ R²⁰¹ R²⁰² R²⁰³ H benzyl isobutyl H; and methyl isobutyl methylisobutyl.Exemplary Amino Acid units include, but are not limited to, units offormula (VII) where: R²⁰⁰ is benzyl and R²⁰¹ is —(CH₂)₄NH₂; R²⁰⁰isopropyl and R²⁰¹ is —(CH₂)₄NH₂; R²⁰⁰ isopropyl and R²⁰¹ is—(CH₂)₃NHCONH₂. Another exemplary Amino Acid unit is a unit of formula(VIII) wherein R²⁰⁰ is benzyl, R²⁰¹ is benzyl, and R²⁰² is —(CH₂)₄NH₂.

Useful —W_(w)— units can be designed and optimized in their selectivityfor enzymatic cleavage by a particular enzymes, for example, atumor-associated protease. In one embodiment, a —W_(w)— unit is thatwhose cleavage is catalyzed by cathepsin B, C and D, or a plasminprotease.

In one embodiment, —W_(w)— is a dipeptide, tripeptide, tetrapeptide orpentapeptide.

When R¹⁹⁰, R²⁰⁰, R²⁰¹, R²⁰² or R²⁰³ is other than hydrogen, the carbonatom to which R¹⁹⁰, R²⁰⁰, R²⁰¹, R²⁰² or R²⁰³ is attached is chiral.

Each carbon atom to which R¹⁹⁰, R²⁰⁰, R²⁰¹, R²⁰² or R²⁰³ is attached isindependently in the (S) or (R) configuration.

In one aspect of the Amino Acid unit, the Amino Acid unit isvaline-citrulline. In another aspect, the Amino Acid unit isphenylalanine-lysine (i.e. fk). In yet another aspect of the Amino Acidunit, the Amino Acid unit is 5-aminovaleric acid,homophenylalanine-lysine, tetraisoquinolinecarboxylate-lysine,cyclohexylalanine-lysine, isonepecotic acid-lysine, beta-alanine-lysine,glycine-serine-valine-glutamine (SEQ ID NO:1) or isonepecotic acid.

In certain embodiments, the Amino Acid unit can comprise natural aminoacids. In other embodiments, the Amino Acid unit can comprisenon-natural amino acids.

The Spacer Unit

The Spacer unit (—Y—), when present, links an Amino Acid unit to theDrug moiety when an Amino Acid unit is present. Alternately, the Spacerunit links the Stretcher unit to the Drug moiety when the Amino Acidunit is absent. The Spacer unit also links the Drug moiety to the Ligandunit when both the Amino Acid unit and Stretcher unit are absent.

Spacer units are of two general types: self-immolative and nonself-immolative. A non self-immolative Spacer unit is one in which partor all of the Spacer unit remains bound to the Drug moiety aftercleavage, particularly enzymatic, of an Amino Acid unit from theDrug-Linker-Ligand Conjugate or the Drug-Linker Compound. Examples of anon self-immolative Spacer unit include, but are not limited to a(glycine-glycine) Spacer unit and a glycine Spacer unit (both depictedin Scheme 1) (infra). When an Exemplary Compound containing aglycine-glycine Spacer unit or a glycine Spacer unit undergoes enzymaticcleavage via a tumor-cell associated-protease, a cancer-cell-associatedprotease or a lymphocyte-associated protease, a glycine-glycine-Drugmoiety or a glycine-Drug moiety is cleaved from L-A_(a)-W_(w)—. In oneembodiment, an independent hydrolysis reaction takes place within thetarget cell, cleaving the glycine-Drug moiety bond and liberating theDrug.

In one embodiment, a non self-immolative Spacer unit (—Y—) is -Gly-Gly-.In another embodiment, a non self-immolative the Spacer unit (—Y—) is-Gly-.

In another embodiment, —Y_(y)— is a p-aminobenzyl alcohol (PAB) unit(see Schemes 2 and 3, infra) whose phenylene portion is substituted withQ_(m) wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or-cyano; and m is an integer ranging from 0-4.

In one embodiment, a Drug-Linker Compound or a Drug-Linker LigandConjugate is provided in which the Spacer unit is absent (y=0), or apharmaceutically acceptable salt or solvate thereof.

Alternatively, an Exemplary Compound containing a self-immolative Spacerunit can release -D without the need for a separate hydrolysis step. Inthis embodiment, —Y— is a PAB group that is linked to —W_(w)— via theamino nitrogen atom of the PAB group, and connected directly to -D via acarbonate, carbamate or ether group. Without being bound by anyparticular theory or mechanism, Scheme 2 depicts a possible mechanism ofDrug release of a PAB group which is attached directly to -D via acarbamate or carbonate group espoused by Toki et al, 2002, J. Org. Chem.67:1866-1872.

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;m is an integer ranging from 0-4; and p is an integer of from 1 to 20.

Without being bound by any particular theory or mechanism, Scheme 3depicts a possible mechanism of Drug release of a PAB group which isattached directly to -D via an ether or amine linkage.

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;m is an integer ranging from 0-4; and p is an integer of from 1 to about20.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB groupsuch as 2-aminoimidazol-5-methanol derivatives (see, e.g., Hay et al.,1999, Bioorg. Med. Chem. Lett. 9:2237) and ortho orpara-aminobenzylacetals. Spacers can be used that undergo cyclizationupon amide bond hydrolysis, such as substituted and unsubstituted4-aminobutyric acid amides (see, e.g., Rodrigues et al., 1995, ChemistryBiology 2:223), appropriately substituted bicyclo[2.2.1] andbicyclo[2.2.2] ring systems (see, e.g., Storm et al., 1972, J. Amer.Chem. Soc. 94:5815) and 2-aminophenylpropionic acid amides (see, e.g.,Amsberry et al., 1990, J. Org. Chem. 55:5867). Elimination ofamine-containing drugs that are substituted at the α-position of glycine(see, e.g., Kingsbury et al., 1984, J. Med. Chem. 27:1447) are alsoexamples of self-immolative spacer useful in Exemplary Compounds.

In one embodiment, the Spacer unit is a branchedbis(hydroxymethyl)styrene (BHMS) unit as depicted in Scheme 4, which canbe used to incorporate and release multiple drugs.

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;m is an integer ranging from 0-4; n is 0 or 1; and p ranges raging from1 to about 20.

In one embodiment, the -D moieties are the same. In yet anotherembodiment, the -D moieties are different.

In one aspect, Spacer units (—Y_(y)—) are represented by Formulas(X)-(XII):

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;and m is an integer ranging from 0-4;

Embodiments of the Formula Ia′ Antibody-Drug Conjugate compoundsinclude:

wherein w and y are each 0,

The drug moiety (D) is of the dolastatin/auristatin type (see, e.g.,U.S. Pat. Nos. 5,635,483; and 5,780,588) which have been shown tointerfere with microtubule dynamics, GTP hydrolysis, and nuclear andcellular division (see, Woyke et al., 2001, Antimicrob. Agents andChemother. 45(12):3580-3584) and have anticancer (see, e.g., U.S. Pat.No. 5,663,149) activity. Some dolastins have antifungal activity (see,e.g., Pettit et al., 1998, Antimicrob. Agents Chemother. 42:2961-2965)

As noted above, D refers to a Drug Unit (moiety) having a nitrogen atomor other atom that can form a bond with the Spacer unit when y=1 or 2,with the C-terminal carboxyl group of an Amino Acid unit when y=0, withthe of a Stretcher unit when w and y=0, and with the Reactive Site of aLigand unit when a, w, and y=0. It is to be understood that the terms“Drug unit” and “Drug moiety” are synonymous and used interchangeablyherein.

In one embodiment, -D is formula D:

-   -   wherein, independently at each location:    -   R² is selected from the group consisting of H and C₁-C₈ alkyl;    -   R³ is selected from the group consisting of H, C₁-C₈ alkyl,        C₃-C₈ carbocycle, aryl, C₁-C₈ alkyl-aryl, X¹—(C₃-C₈ carbocycle),        C₃-C₈ heterocycle and X¹—(C₃-C₈ heterocycle);    -   R⁴ is selected from the group consisting of H, C₁-C₈ alkyl,        C₃-C₈ carbocycle, aryl, X¹-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle),        C₃-C₈ heterocycle and X¹—(C₃-C₈ heterocycle);    -   R⁵ is selected from the group consisting of H and methyl;    -   or R⁴ and R⁵ jointly form a carbocyclic ring and have the        formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from the group consisting of H, C₁-C₈        alkyl and C₃-C₈ carbocycle and n is selected from the group        consisting of 2, 3, 4, 5 and 6;    -   R⁶ is selected from the group consisting of H and C₁-C₈ alkyl;    -   R⁷ is selected from the group consisting of H, C₁-C₈ alkyl,        C₃-C₈ carbocycle, aryl, X¹-aryl, X¹—(C₃-C₈ carbocycle), C₃-C₈        heterocycle and X¹—(C₃-C₈ heterocycle);    -   each R⁸ is independently selected from the group consisting of        H, OH, C₁-C₈ alkyl, C₃-C₈ carbocycle and O—(C₁-C₈ alkyl);    -   each X¹ is independently C₁-C₁₀ alkylene; and    -   the moiety —NR⁹Z¹ is a phenylalanine bioisostere of any of the        above embodiments.

In one embodiment, R³, R⁴ and R⁷ are independently isopropyl orsec-butyl and R⁵ is —H. In an exemplary embodiment, R³ and R⁴ are eachisopropyl, R⁵ is H, and R⁷ is sec-butyl.

In another embodiment, R² and R⁶ are each methyl, and R⁹ is H.

In still another embodiment, each occurrence of R⁸ is —OCH₃.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R² and R⁶ areeach methyl, R⁵ is H, R⁷ is sec-butyl, each occurrence of R⁸ is —OCH₃,and R⁹ is H.

Illustrative Drug units (-D) include the drug units have the followingstructure:

and pharmaceutically acceptable salts or solvates thereof wherein R¹⁰and Z² have the meanings provided above.

In one aspect, hydrophilic groups, such as but not limited totriethylene glycol esters (TEG) can be attached to the Drug Unit.Without being bound by theory, the hydrophilic groups assist in theinternalization and non-agglomeration of the Drug Unit.

The Ligand Unit (L)

The Ligand unit (L-) includes within its scope any unit of a Ligand (L)that binds or reactively associates or complexes with a receptor,antigen or other receptive moiety associated with a given target-cellpopulation. A Ligand unit is a molecule that binds to, complexes with,or reacts with a receptor, antigen or other receptive moiety of a cellpopulation sought to be therapeutically or otherwise biologicallymodified. In one aspect, the Ligand unit acts to deliver the Drug unitto the particular target cell population with which the Ligand unitinteracts. Such Ligands include, but are not limited to, large molecularweight proteins such as, for example, full-length antibodies, antibodyfragments, smaller molecular weight proteins, polypeptide or peptides,lectins, glycoproteins, non-peptides, vitamins, nutrient-transportmolecules (such as, but not limited to, transferrin), or any other cellbinding molecule or substance.

A Ligand unit can form a bond to a Stretcher unit, an Amino Acid unit, aSpacer Unit, or a Drug Unit. A Ligand unit can form a bond to a Linkerunit via a heteroatom of the Ligand. Heteroatoms that may be present ona Ligand unit include sulfur (in one embodiment, from a sulfhydryl groupof a Ligand), oxygen (in one embodiment, from a carbonyl, carboxyl orhydroxyl group of a Ligand) and nitrogen (in one embodiment, from aprimary or secondary amino group of a Ligand). These heteroatoms can bepresent on the Ligand in the Ligand's natural state, for example anaturally-occurring antibody, or can be introduced into the Ligand viachemical modification.

In one embodiment, a Ligand has a sulfhydryl group and the Ligand bondsto the Linker unit via the sulfhydryl group's sulfur atom.

In another embodiment, the Ligand has lysine residues that can reactwith activated esters (such esters include, but are not limited to,N-hydroxysuccinimde, pentafluorophenyl, and p-nitrophenyl esters) of theLinker and thus form an amide bond consisting of the nitrogen atom ofthe Ligand and the C═O group of the Linker.

In yet another aspect, the Ligand has one or more lysine residues thatcan be chemically modified to introduce one or more sulfhydryl groups.The Ligand unit bonds to the Linker unit via the sulfhydryl group'ssulfur atom. The reagents that can be used to modify lysines include,but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and2-Iminothiolane hydrochloride (Traut's Reagent).

In another embodiment, the Ligand can have one or more carbohydrategroups that can be chemically modified to have one or more sulfhydrylgroups. The Ligand unit bonds to the Linker unit, such as the StretcherUnit, via the sulfhydryl group's sulfur atom.

In yet another embodiment, the Ligand can have one or more carbohydrategroups that can be oxidized to provide an aldehyde (—CHO) group (see,e.g., Laguzza, et al., 1989, J. Med. Chem. 32(3):548-55). Thecorresponding aldehyde can form a bond with a Reactive Site on aStretcher. Reactive sites on a Stretcher that can react with a carbonylgroup on a Ligand include, but are not limited to, hydrazine andhydroxylamine. Other protocols for the modification of proteins for theattachment or association of Drug Units are described in Coligan et al.,Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002)(incorporated herein by reference).

Useful non-immunoreactive protein, polypeptide, or peptide Ligandsinclude, but are not limited to, transferrin, epidermal growth factors(“EGF”), bombesin, gastrin, gastrin-releasing peptide, platelet-derivedgrowth factor, IL-2, IL-6, transforming growth factors (“TGF”), such asTGF-α and TGF-β, vaccinia growth factor (“VGF”), insulin andinsulin-like growth factors I and II, somatostatin, lectins andapoprotein from low density lipoprotein.

Useful polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of immunized animals. Useful monoclonalantibodies are homogeneous populations of antibodies to a particularantigenic determinant (e.g., a cancer cell antigen, a viral antigen, amicrobial antigen, a protein, a peptide, a carbohydrate, a chemical,nucleic acid, or fragments thereof). A monoclonal antibody (mAb) to anantigen-of-interest can be prepared by using any technique known in theart which provides for the production of antibody molecules bycontinuous cell lines in culture.

Useful monoclonal antibodies include, but are not limited to, humanmonoclonal antibodies, humanized monoclonal antibodies, antibodyfragments, or chimeric human-mouse (or other species) monoclonalantibodies. Human monoclonal antibodies may be made by any of numeroustechniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad.Sci. USA. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79;and Olsson et al., 1982, Meth. Enzymol. 92:3-16).

The antibody can also be a bispecific antibody. Methods for makingbispecific antibodies are known in the art and are discussed infra.

The antibody can be a functionally active fragment, derivative or analogof an antibody that immunospecifically binds to target cells (e.g.,cancer cell antigens, viral antigens, or microbial antigens) or otherantibodies bound to tumor cells or matrix. In this regard, “functionallyactive” means that the fragment, derivative or analog is able to elicitanti-anti-idiotype antibodies that recognize the same antigen that theantibody from which the fragment, derivative or analog is derivedrecognized. Specifically, in an exemplary embodiment the antigenicity ofthe idiotype of the immunoglobulin molecule can be enhanced by deletionof framework and CDR sequences that are C-terminal to the CDR sequencethat specifically recognizes the antigen. To determine which CDRsequences bind the antigen, synthetic peptides containing the CDRsequences can be used in binding assays with the antigen by any bindingassay method known in the art (e.g., the BIA core assay) (See, e.g.,Kabat et al., 1991, Sequences of Proteins of Immunological Interest,Fifth Edition, National Institute of Health, Bethesda, Md.; Kabat E etal., 1980, J. Immunology 125(3):961-969).

Other useful antibodies include fragments of antibodies such as, but notlimited to, F(ab′)₂ fragments, Fab fragments, Fvs, single chainantibodies, diabodies, tribodies, tetrabodies, scFv, scFv-FV, or anyother molecule with the same specificity as the antibody.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are usefulantibodies. A chimeric antibody is a molecule in which differentportions are derived from different animal species, such as for example,those having a variable region derived from a murine monoclonal andhuman immunoglobulin constant regions. (See, e.g., U.S. Pat. No.4,816,567; and U.S. Pat. No. 4,816,397, which are incorporated herein byreference in their entirety.) Humanized antibodies are antibodymolecules from non-human species having one or more complementaritydetermining regions (CDRs) from the non-human species and a frameworkregion from a human immunoglobulin molecule. (See, e.g., U.S. Pat. No.5,585,089, which is incorporated herein by reference in its entirety.)Such chimeric and humanized monoclonal antibodies can be produced byrecombinant DNA techniques known in the art, for example using methodsdescribed in International Publication No. WO 87/02671; European PatentPublication No. 0 184 187; European Patent Publication No. 0 171 496;European Patent Publication No. 0 173 494; International Publication No.WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Publication No.012 023; Berter et al., 1988, Science 240:1041-1043; Liu et al., 1987,Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol.139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-218;Nishimura et al., 1987, Cancer. Res. 47:999-1005; Wood et al., 1985,Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst.80:1553-1559; Morrison, 1985, Science 229:1202-1207; Oi et al., 1986,BioTechniques 4:214; U.S. Pat. No. 5,225,539; Jones et al., 1986, Nature321:552-525; Verhoeyan et al., 1988, Science 239:1534; and Beidler etal., 1988, J. Immunol. 141:4053-4060; each of which is incorporatedherein by reference in its entirety.

Completely human antibodies are particularly desirable and can beproduced using transgenic mice that are incapable of expressingendogenous immunoglobulin heavy and light chains genes, but which canexpress human heavy and light chain genes. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained using conventionalhybridoma technology. The human immunoglobulin transgenes harbored bythe transgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgA, IgM and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg and Huszar, 1995, Int. Rev.Immunol. 13:65-93). For a detailed discussion of this technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies. See, e.g., U.S. Pat. Nos. 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; each of which isincorporated herein by reference in its entirety. Other human antibodiescan be obtained commercially from, for example, Abgenix, Inc. (nowAmgen, Freemont, Calif.) and Medarex (Princeton, N.J.).

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (See, e.g., Jespers et al., 1994,Biotechnology 12:899-903). Human antibodies can also be produced usingvarious techniques known in the art, including phage display libraries(see, e.g., Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381; Marks etal., 1991, J. Mol. Biol. 222:581; Quan and Carter, 2002, The rise ofmonoclonal antibodies as therapeutics, In Anti-IgE and Allergic Disease,Jardieu and Fick, eds., Marcel Dekker, New York, N.Y., Chapter 20, pp.427-469).

In other embodiments, the antibody is a fusion protein of an antibody,or a functionally active fragment thereof, for example in which theantibody is fused via a covalent bond (e.g., a peptide bond), at eitherthe N-terminus or the C-terminus to an amino acid sequence of anotherprotein (or portion thereof, preferably at least 10, 20 or 50 amino acidportion of the protein) that is not from an antibody. Preferably, theantibody or fragment thereof is covalently linked to the other proteinat the N-terminus of the constant domain.

Antibodies include analogs and derivatives that are either modified,i.e., by the covalent attachment of any type of molecule as long as suchcovalent attachment permits the antibody to retain its antigen bindingimmunospecificity. For example, but not by way of limitation,derivatives and analogs of the antibodies include those that have beenfurther modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular antibody unit orother protein, etc. Any of numerous chemical modifications can becarried out by known techniques including, but not limited to, specificchemical cleavage, acetylation, formylation, metabolic synthesis in thepresence of tunicamycin, etc. Additionally, the analog or derivative cancontain one or more unnatural amino acids.

Antibodies can have modifications (e.g., substitutions, deletions oradditions) in amino acid residues that interact with Fc receptors. Inparticular, antibodies can have modifications in amino acid residuesidentified as involved in the interaction between the anti-Fc domain andthe FcRn receptor (see, e.g., International Publication No. WO 97/34631,which is incorporated herein by reference in its entirety).

Antibodies immunospecific for a cancer cell antigen can be obtainedcommercially or produced by any method known to one of skill in the artsuch as, e.g., chemical synthesis or recombinant expression techniques.The nucleotide sequence encoding antibodies immunospecific for a cancercell antigen can be obtained, e.g., from the GenBank database or adatabase like it, the literature publications, or by routine cloning andsequencing.

In a specific embodiment, known antibodies for the treatment orprevention of cancer can be used. Antibodies immunospecific for a cancercell antigen can be obtained commercially or produced by any methodknown to one of skill in the art such as, e.g., recombinant expressiontechniques. The nucleotide sequence encoding antibodies immunospecificfor a cancer cell antigen can be obtained, e.g., from the GenBankdatabase or a database like it, the literature publications, or byroutine cloning and sequencing. Examples of antibodies available for thetreatment of cancer include, but are not limited to, RITUXAN®(rituximab; Genentech) which is a chimeric anti-CD20 monoclonal antibodyfor the treatment of patients with non-Hodgkin's lymphoma; OVAREX whichis a murine antibody for the treatment of ovarian cancer; PANOREX (GlaxoWellcome, NC) which is a murine IgG_(2a) antibody for the treatment ofcolorectal cancer; Cetuximab ERBITUX (Imclone Systems Inc., NY) which isan anti-EGFR IgG chimeric antibody for the treatment of epidermal growthfactor positive cancers, such as head and neck cancer; Vitaxin(MedImmune, Inc., MD) which is a humanized antibody for the treatment ofsarcoma; CAMPATH I/H (Leukosite, MA) which is a humanized IgG₁ antibodyfor the treatment of chronic lymphocytic leukemia (CLL); SMART MI195(Protein Design Labs, Inc., CA) and SGN-33 (Seattle Genetics, Inc., WA)which is a humanized anti-CD33 IgG antibody for the treatment of acutemyeloid leukemia (AML); LYMPHOCIDE (Immunomedics, Inc., NJ) which is ahumanized anti-CD22 IgG antibody for the treatment of non-Hodgkin'slymphoma; SMART ID10 (Protein Design Labs, Inc., CA) which is ahumanized anti-HLA-DR antibody for the treatment of non-Hodgkin'slymphoma; ONCOLYM (Techniclone, Inc., CA) which is a radiolabeled murineanti-HLA-Dr10 antibody for the treatment of non-Hodgkin's lymphoma;ALLOMUNE (BioTransplant, CA) which is a humanized anti-CD2 mAb for thetreatment of Hodgkin's Disease or non-Hodgkin's lymphoma; AVASTIN(Genentech, Inc., CA) which is an anti-VEGF humanized antibody for thetreatment of lung and colorectal cancers; EPRATUZAMAB (Immunomedics,Inc., NJ and Amgen, CA) which is an anti-CD22 antibody for the treatmentof non-Hodgkin's lymphoma; and CEACIDE (Immunomedics, NJ) which is ahumanized anti-CEA antibody for the treatment of colorectal cancer.

Other antibodies useful in the treatment of cancer include, but are notlimited to, antibodies against the following antigens (where exemplarycancers that can be treated with the antibody are in parentheses): CA125(ovarian), CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas),Lewis Y (carcinomas), Lewis X (carcinomas), alpha fetoprotein(carcinomas), CA 242 (colorectal), placental alkaline phosphatase(carcinomas), prostate specific antigen (prostate), prostate specificmembrane antigen (prostate), prostatic acid phosphatase (prostate),epidermal growth factor (carcinomas), MAGE-1 (carcinomas), MAGE-2(carcinomas), MAGE-3 (carcinomas), MAGE-4 (carcinomas), anti-transferrinreceptor (carcinomas), p97 (melanoma), MUC1-KLH (breast cancer), CEA(colorectal), gp100 (melanoma), MART1 (melanoma), IL-2 receptor (T-cellleukemia and lymphomas), CD20 (non-Hodgkin's lymphoma), CD52 (leukemia),CD33 (leukemia), CD22 (lymphoma), human chorionic gonadotropin(carcinoma), CD38 (multiple myeloma), CD40 (lymphoma), mucin(carcinomas), P21 (carcinomas), MPG (melanoma), and Neu oncogene product(carcinomas). Some specific, useful antibodies include, but are notlimited to, BR96 mAb (Trail et al., 1993, Science 261:212-215), BR64(Trail et al., 1997, Cancer Research 57:100-105), mAbs against the CD40antigen, such as S2C6 mAb (Francisco et al., 2000, Cancer Res.60:3225-3231), mAbs against the CD70 antigen, such as 1F6 mAb, humanized1F6 mAb, 2F2 mAb and humanized 2F2 mAb (see, e.g., InternationalPublished Application No. WO 04/073656 and U.S. Published ApplicationNo. 2006-0083736) and mAbs against the CD30 antigen, such as AC10 (Bowenet al., 1993, J. Immunol. 151:5896-5906; Wahl et al., 2002 Cancer Res.62(13):3736-42) and MDX-060. Many other internalizing antibodies thatbind to tumor associated antigens can be used and have been reviewed(see, e.g., Franke et al., 2000, Cancer Biother. Radiopharm. 15, 459-76;Murray, 2000, Semin Oncol. 27:64-70; Breitling and Dubel, RecombinantAntibodies, John Wiley, and Sons, New York, 1998).

In another specific embodiment, antibodies for the treatment orprevention of an autoimmune disease are used in accordance with thecompositions and methods of the invention. Antibodies immunospecific foran antigen of a cell that is responsible for producing autoimmuneantibodies can be obtained from any organization (e.g., a universityscientist or a company) or produced by any method known to one of skillin the art such as, e.g., chemical synthesis or recombinant expressiontechniques. In another embodiment, useful antibodies are immunospecificfor the treatment of autoimmune diseases include, but are not limitedto, anti-nuclear antibody; anti-ds DNA; Anti-ss DNA, anti-cardiolipinantibody IgM, IgG; anti-phospholipid antibody IgM, IgG; anti-SMantibody; anti-mitochondrial antibody; thyroid antibody; microsomalantibody; thyroglobulin antibody; anti-SCL-70 antibody; anti-Joantibody; anti-U₁RNP antibody; anti-La/SSB antibody; anti-SSA; anti-SSBantibody; anti-perital cells antibody; anti-histones antibody; anti-RNPantibody; C-ANCA antibody; P-ANCA antibody; anti-centromere antibody;Anti-Fibrillarin antibody and anti-GBM antibody.

In certain embodiments, useful antibodies can bind to a receptor or areceptor complex expressed on an activated lymphocyte. The receptor orreceptor complex can comprise an immunoglobulin gene superfamily member,a TNF receptor superfamily member, an integrin, a cytokine receptor, achemokine receptor, a major histocompatibility protein, a lectin, or acomplement control protein. Non-limiting examples of suitableimmunoglobulin superfamily members are CD2, CD3, CD4, CD8, CD19, CD22,CD28, CD79, CD90, CD152/CTLA-4, PD-1, and ICOS. Non-limiting examples ofsuitable TNF receptor superfamily members are CD27, CD40, CD95/Fas,CD134/OX40, CD137/4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA,osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3.Non-limiting examples of suitable integrins are CD11a, CD11b, CD11c,CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103, andCD104. Non-limiting examples of suitable lectins are C-type, S-type, andI-type lectin.

In one embodiment, the Ligand unit binds to an activated lymphocyte thatis associated with an autoimmune disease.

In another specific embodiment, useful Ligands immunospecific for aviral or a microbial antigen are monoclonal antibodies. The antibodiesmay be chimeric, humanized or human monoclonal antibodies. As usedherein, the term “viral antigen” includes, but is not limited to, anyviral peptide, polypeptide protein (e.g., HIV gp120, HIV nef, RSV Fglycoprotein, influenza virus neuraminidase, influenza virushemagglutinin, HTLV tax, herpes simplex virus glycoprotein (e.g., gB,gC, gD, and gE) and hepatitis B surface antigen) that is capable ofeliciting an immune response. As used herein, the term “microbialantigen” includes, but is not limited to, any microbial peptide,polypeptide, protein, saccharide, polysaccharide, or lipid molecule(e.g., a bacterial, fungi, pathogenic protozoa, or yeast polypeptideincluding, e.g., LPS and capsular polysaccharide 5/8) that is capable ofeliciting an immune response.

Antibodies immunospecific for a viral or microbial antigen can beobtained commercially, for example, from BD Biosciences (San Francisco,Calif.), Chemicon International, Inc. (Temecula, Calif.), or VectorLaboratories, Inc. (Burlingame, Calif.) or produced by any method knownto one of skill in the art such as, e.g., chemical synthesis orrecombinant expression techniques. The nucleotide sequence encodingantibodies that are immunospecific for a viral or microbial antigen canbe obtained, e.g., from the GenBank database or a database like it,literature publications, or by routine cloning and sequencing.

In a specific embodiment, useful Ligands are those that are useful forthe treatment or prevention of viral or microbial infection inaccordance with the methods disclosed herein. Examples of antibodiesavailable useful for the treatment of viral infection or microbialinfection include, but are not limited to, SYNAGIS (MedImmune, Inc., MD)which is a humanized anti-respiratory syncytial virus (RSV) monoclonalantibody useful for the treatment of patients with RSV infection; PRO542(Progenics) which is a CD4 fusion antibody useful for the treatment ofHIV infection; OSTAVIR (Protein Design Labs, Inc., CA) which is a humanantibody useful for the treatment of hepatitis B virus; PROTOVIR(Protein Design Labs, Inc, CA) which is a humanized IgG₁ antibody usefulfor the treatment of cytomegalovirus (CMV); and anti-LPS antibodies.

Other antibodies useful in the treatment of infectious diseases include,but are not limited to, antibodies against the antigens from pathogenicstrains of bacteria (Streptococcus pyogenes, Streptococcus pneumoniae,Neisseria gonorrheae, Neisseria meningitidis, Corynebacteriumdiphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridiumtetani, Hemophilus influenzae, Klebsiella pneumoniae, Klebsiellaozaenas, Klebsiella rhinoscleromotis, Staphylococc aureus, Vibriocolerae, Escherichia coli, Pseudoinonas aeraginosa, Campylobacter(Vibrio) fetus, Aeromonas hydrophila, Bacillus cereus, Edwardsiellatarda, Yersinia enterocolitica, Yersinia pestis, Yersiniapseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigellasonnei, Salmonella typhimurium, Treponema pallidum, Treponema pertenue,Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi,Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Pneumocystiscarinii, Francisella tularensis, Brucella abortus, Brucella suis,Brucella melitensis, Mycoplasnia spp., Rickettsia prowazeki, Rickettsiatsutsugumushi, and Chlamydia spp.); pathogenic fungi (Coccidioidesimmitis, Aspergillus fumigatus, Candida albicans, Blastomycesdermatitidis, Cryptococcus neoformans, and Histoplasma capsulatum);protozoa (Entomoeba histolytica, Toxoplasma gondii, Trichomonas tenas,Trichomonas hominis, Trichomonas vaginalis, Trypanosoma gambiense,Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani,Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia,Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); orHelminiths (Enterobius vermicularis, Trichuris trichiura, Ascarislumbricoides, Trichinella spiralis, Strongyloides stercoralis,Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, andhookworms).

Other antibodies useful in this invention for treatment of viral diseaseinclude, but are not limited to, antibodies against antigens ofpathogenic viruses, including as examples and not by limitation:Poxyiridae, Herpesviridae, Herpes Simplex virus 1, Herpes Simplex virus2, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae,Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenzaviruses, mumps, measles, respiratory syncytial virus, rubella,Arboviridae, Rhabdoviridae, Arenaviridae, Hepatitis A virus, Hepatitis Bvirus, Hepatitis C virus, Hepatitis E virus, Non-A/Non-B Hepatitisvirus, Rhinoviridae, Coronaviridae, Rotoviridae, and HumanImmunodeficiency Virus.

In attempts to discover effective cellular targets for cancer diagnosisand therapy, researchers have sought to identify transmembrane orotherwise tumor-associated polypeptides that are specifically expressedon the surface of one or more particular type(s) of cancer cell ascompared to on one or more normal non-cancerous cell(s). Often, suchtumor-associated polypeptides are more abundantly expressed on thesurface of the cancer cells as compared to on the surface of thenon-cancerous cells. The identification of such tumor-associated cellsurface antigen polypeptides has given rise to the ability tospecifically target cancer cells for destruction via antibody-basedtherapies.

In an exemplary embodiment, the Ligand-Linker-Drug Conjugate has FormulaIIIa, where the Ligand is an antibody Ab that binds at least one ofCD20, CD30, CD33, CD40, CD70, BCMA, and Lewis Y antigen, w=0, y=0, and Dhas Formula Ib. Exemplary Conjugates of Formula IIIa include those inwhich R¹⁷ is —(CH₂)₅—. Also included are such Conjugates of Formula IIIacontaining about 2 to about 8, or about 2 to about 6 Drug moieties D perLigand unit (that is, Conjugates of Formula Ia wherein p is a value inthe range about 2-8, for example about 2-6). Conjugates containingcombinations of the structural features noted in this paragraph are alsocontemplated as within the scope of the compounds of the invention.

In another embodiment, the Ligand-Linker-Drug Conjugate has FormulaIIIa, where Ligand is an Antibody Ab that binds one of CD20, CD30, CD33,CD40, CD70, BCMA, and Lewis Y antigen, w=1, y=0, and D has Formula Ib.Included are such Conjugates of Formula IIIa in which R¹⁷ is —(CH₂)₅—.Also included are such Conjugates of Formula IIIa containing about 2 toabout 8, or about 2 to about 6 Drug moieties D per Ligand unit (that is,Conjugates of Formula Ia wherein p is a value in the range of about 2-8,or about 2-6). Conjugates containing combinations of the structuralfeatures noted in this paragraph are also exemplary.

In another embodiment, the Ligand-Linker-Drug Conjugate has FormulaIIIa, where the Ligand is an Antibody Ab that binds one of CD20, CD30,CD33, CD40, CD70, BCMA, and Lewis Y antigen, w=1, y=1, and D has FormulaIb. Included are Conjugates of Formula IIIa in which R¹⁷ is —(CH₂)₅—.Conjugates containing combinations of the structural features noted inthis paragraph are also contemplated within the scope of the compoundsof the invention.

Production of Recombinant Antibodies

Antibodies of the invention can be produced using any method known inthe art to be useful for the synthesis of antibodies, in particular, bychemical synthesis or by recombinant expression.

Recombinant expression of antibodies, or fragment, derivative or analogthereof, requires construction of a nucleic acid that encodes theantibody. If the nucleotide sequence of the antibody is known, a nucleicacid encoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., 1994,BioTechniques 17:242), which involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligation of those oligonucleotides, and thenamplification of the ligated oligonucleotides, e.g., by PCR.

Alternatively, a nucleic acid molecule encoding an antibody can begenerated from a suitable source. If a clone containing the nucleic acidencoding the particular antibody is not available, but the sequence ofthe antibody is known, a nucleic acid encoding the antibody can beobtained from a suitable source (e.g., an antibody cDNA library, or cDNAlibrary generated from any tissue or cells expressing theimmunoglobulin) by, e.g., PCR amplification using synthetic primershybridizable to the 3′ and 5′ ends of the sequence or by cloning usingan oligonucleotide probe specific for the particular gene sequence.

If an antibody that specifically recognizes a particular antigen is notcommercially available (or a source for a cDNA library for cloning anucleic acid encoding such an immunoglobulin), antibodies specific for aparticular antigen can be generated by any method known in the art, forexample, by immunizing a non-human animal, or suitable animal model suchas a rabbit or mouse, to generate polyclonal antibodies or, morepreferably, by generating monoclonal antibodies, e.g., as described byKohler and Milstein (1975, Nature 256:495-497) or as described by Kozboret al. (1983, Immunology Today 4:72) or Cole et al. (1985 in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).Alternatively, a clone encoding at least the Fab portion of the antibodycan be obtained by screening Fab expression libraries (e.g., asdescribed in Huse et al., 1989, Science 246:1275-1281) for clones of Fabfragments that bind the specific antigen or by screening antibodylibraries (see, e.g., Clackson et al., 1991, Nature 352:624; Hane etal., 1997, Proc. Natl. Acad. Sci. USA 94:4937).

Once a nucleic acid sequence encoding at least the variable domain ofthe antibody is obtained, it can be introduced into a vector containingthe nucleotide sequence encoding the constant regions of the antibody(see, e.g., International Publication No. WO 86/05807; WO 89/01036; andU.S. Pat. No. 5,122,464). Vectors containing the complete light or heavychain that allow for the expression of a complete antibody molecule areavailable. The nucleic acid encoding the antibody can be used tointroduce the nucleotide substitutions or deletion necessary tosubstitute (or delete) the one or more variable region cysteine residuesparticipating in an intrachain disulfide bond with an amino acid residuethat does not contain a sulfhydryl group. Such modifications can becarried out by any method known in the art for the introduction ofspecific mutations or deletions in a nucleotide sequence, for example,but not limited to, chemical mutagenesis and in vitro site directedmutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253:6551).

In addition, techniques developed for the production of “chimericantibodies” (see, e.g., Morrison et al., 1984, Proc. Natl. Acad. Sci.81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al.,1985, Nature 314:452-454) by splicing genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine monoclonal antibody and a humanimmunoglobulin constant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-54) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,1988, Science 242:1038-1041).

Antibody fragments that recognize specific epitopes can be generated byknown techniques. For example, such fragments include F(ab′)₂ fragments,Fab fragments, Fv fragments, diabodies, triabodies, tetrabodies, singlechain antibodies, scFv, scFv-Fc and the like.

Once a nucleic acid sequence encoding an antibody has been obtained, thevector for the production of the antibody can be produced by recombinantDNA technology using techniques well known in the art. Methods that arewell known to those skilled in the art can be used to constructexpression vectors containing the antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. See, forexample, the techniques described in Sambrook et al. (1990, MolecularCloning, A Laboratory Manual, 2^(nd) Ed., Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.; 2001; Molecular Cloning, A Laboratory Manual,3^(rd) ed., Cold Spring Harbor Publish., Cold Spring Harbor, N.Y.) andAusubel et al., eds., in Current Protocols in Molecular Biology seriesof laboratory technique manuals, 1987-1999, Current Protocols,© 1994-199John Wiley and Sons, Inc.).

An expression vector comprising the nucleotide sequence of an antibodyor the nucleotide sequence of an antibody can be transferred to a hostcell by conventional techniques (e.g., electroporation, liposomaltransfection, and calcium phosphate precipitation), and the transfectedcells are then cultured by conventional techniques to produce theantibody. In specific embodiments, the expression of the antibody isregulated by a constitutive, an inducible or a tissue, specificpromoter.

The host cells used to express the recombinant antibody can be eitherbacterial cells such as Escherichia coli or eukaryotic cells, especiallyfor the expression of whole recombinant immunoglobulin molecule. Inparticular, mammalian cells such as Chinese hamster ovary cells (CHO),in conjunction with a vector such as the major intermediate early genepromoter element from human cytomegalovirus is an effective expressionsystem for immunoglobulins (Foecking et al., 198, Gene 45:101; Cockettet al., 1990, BioTechnology 8:2).

A variety of host-expression vector systems can be utilized to expressthe immunoglobulin antibodies. Such host-expression systems representvehicles by which the coding sequences of the antibody can be producedand subsequently purified, but also represent cells that can, whentransformed or transfected with the appropriate nucleotide codingsequences, express an antibody immunoglobulin molecule in situ. Theseinclude, but are not limited to, microorganisms such as bacteria (e.g.,E. coli and B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing immunoglobulincoding sequences; yeast (e.g., Saccharomyces Pichia) transformed withrecombinant yeast expression vectors containing immunoglobulin codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the immunoglobulincoding sequences; plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus (CaMV) and tobaccomosaic virus (TMV)) or transformed with recombinant plasmid expressionvectors (e.g., Ti plasmid) containing immunoglobulin coding sequences;or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, or 3T3 cells)harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the antibodybeing expressed. For example, when a large quantity of such a protein isto be produced, vectors that direct the expression of high levels offusion protein products that are readily purified might be desirable.Such vectors include, but are not limited, to the E. coli expressionvector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which theantibody coding sequence may be ligated individually into the vector inframe with the lac Z coding region so that a fusion protein is produced;pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; VanHeeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEXVectors can also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption and binding to a matrix glutathione-agarose beads followed byelution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) or the analogous virus from Drosophila Melanogaster can be usedas a vector to express foreign genes. The virus grows in Spodopterafrugiperda cells. The antibody coding sequence can be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) results in a recombinant virus that is viable and capable ofexpressing the immunoglobulin molecule in infected hosts. (e.g., seeLogan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specificinitiation signals can also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression canbe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see, e.g., Bittner et al.,1987, Methods in Enzymol. 153:51-544). In some embodiments, antibodiescan be expressed using the CHEF system. (See, e.g., U.S. Pat. No.5,888,809; the disclosure of which is incorporated by reference herein.)

In addition, a host cell strain can be chosen to modulate the expressionof the inserted sequences, or modifies and processes the gene product inthe specific fashion desired. Such modifications (e.g., glycosylation)and processing (e.g., cleavage) of protein products can be important forthe function of the protein. Different host cells have characteristicand specific mechanisms for the post-translational processing andmodification of proteins and gene products. Appropriate cell lines orhost systems can be chosen to ensure the correct modification andprocessing of the foreign protein expressed. To this end, eukaryotichost cells that possess the cellular machinery for proper processing ofthe primary transcript, glycosylation, and phosphorylation of the geneproduct can be used. Such mammalian host cells include, but are notlimited to, CHO (e.g., DG44), VERY, BH, Hela, COS, MDCK, 293, 293T, 3T3,WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is typically used. For example, cell lines that stablyexpress an antibody can be engineered. Rather than using expressionvectors that contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells can beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci that in turncan be cloned and expanded into cell lines. This method canadvantageously be used to engineer cell lines which express theantibody. Such engineered cell lines can be particularly useful inscreening and evaluation of tumor antigens that interact directly orindirectly with the antibody.

A number of selection systems can be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can beemployed in tk⁻, hgprt⁻ or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: DHFR, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan,1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev.Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215) and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (supra; Kriegler, 1990, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY; and inChapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols inHuman Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981,J. Mol. Biol. 150:1).

The expression levels of an antibody can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing an antibody isamplifiable, an increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the nucleotide sequence of theantibody, production of the antibody will also increase (see, e.g.,Crouse et al., 1983, Mol. Cell. Biol. 3:257).

The host cell can be co-transfected with two expression vectors, thefirst vector encoding a heavy chain derived polypeptide and the secondvector encoding a light chain derived polypeptide. The two vectors cancontain identical selectable markers that enable equal expression ofheavy and light chain polypeptides. Alternatively, a single vector canbe used to encode both heavy and light chain polypeptides. In suchsituations, the light chain is typically placed before the heavy chainto avoid an excess of toxic free heavy chain (see, e.g., Proudfoot,1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197).The coding sequences for the heavy and light chains can comprise cDNA orgenomic DNA.

Once the antibody has been recombinantly expressed, it can be purifiedusing any method known in the art for purification of an antibody, forexample, by chromatography (e.g., ion exchange, affinity, particularlyby affinity for the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.

In yet another exemplary embodiment, the antibody is a monoclonalantibody.

Production of Antibodies

The production of antibodies will be illustrated with reference toanti-CD30 antibodies but it will be apparent for those skilled in theart that antibodies to other targets (such as members of the TNFreceptor family) can be produced and modified in a similar manner. Theuse of CD30 for the production of antibodies is exemplary only and notintended to be limiting.

The CD30 antigen to be used for production of antibodies may be, e.g., asoluble form of the extracellular domain of CD30 or a portion thereof,containing the desired epitope. Alternatively, cells expressing CD30 attheir cell surface (e.g., L540 (Hodgkin's lymphoma derived cell linewith a T cell phenotype) and L428 (Hodgkin's lymphoma derived cell linewith a B cell phenotype)) can be used to generate antibodies. Otherforms of CD30 useful for generating antibodies will be apparent to thoseskilled in the art.

(i) Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Typically, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

(ii) Monoclonal Antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the monoclonal antibodies may be made using the hybridomamethod first described by Kohler et al., 1975, Nature 256:495, or may bemade by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (see, e.g., Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (see, e.g., Kozbor, 1984, J. Immunol. 133:3001; and Brodeuret al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). The binding affinity of the monoclonalantibody can, for example, be determined by the Scatchard analysis ofMunson et al., 1980, Anal. Biochem. 107:220.

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells serveas a preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells,or myeloma cells that do not otherwise produce antibody protein, toobtain the synthesis of monoclonal antibodies in the recombinant hostcells. Review articles on recombinant expression in bacteria of DNAencoding the antibody include Skerra et al., 1993, Curr. Opinion inImmunol. 5:256-262 and Plückthun, 1992, Immunol. Revs. 130:151-188.

In a further embodiment, monoclonal antibodies or antibody fragments canbe isolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., 1990, Nature 348:552-554. Clackson etal., 1991, Nature, 352:624-628 and Marks et al., 1991, J. Mol. Biol.222:581-597 describe the isolation of murine and human antibodies,respectively, using phage libraries. Subsequent publications describethe production of high affinity (nM range) human antibodies by chainshuffling (Marks et al., 1992, Bio/Technology, 10:779-783), as well ascombinatorial infection and in vivo recombination as a strategy forconstructing very large phage libraries (see, e.g., Waterhouse et al.,1993, Nuc. Acids. Res., 21:2265-2266). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy chain and light chain constant domains in placeof the homologous murine sequences (see, e.g., U.S. Pat. No. 4,816,567;and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851), or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

(iii) Humanized Antibodies

A humanized antibody may have one or more amino acid residues introducedinto it from a source which is non-human. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (see, e.g.,Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature332:323-327; Verhoeyen et al., 1988, Science 239:1534-1536), bysubstituting hypervariable region sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (see, e.g., U.S. Pat. No. 4,816,567) whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome hypervariable region residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is important to reduce antigenicity.According to the so-called “best-fit” method, the sequence of thevariable domain of a rodent antibody is screened against the entirelibrary of known human variable-domain sequences. The human sequencewhich is closest to that of the rodent is then accepted as the humanframework region (FR) for the humanized antibody (Sims et al., 1993, J.Immunol. 151:2296; Chothia et al., 1987, J. Mol. Biol. 196:901). Anothermethod uses a particular framework region derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies (see, e.g., Carter et al., 1992, Proc. Natl. Acad.Sci. USA, 89:4285; Presta et al., 1993, J. Immunol. 151:2623).

In another embodiment, the antibodies may be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.Humanized antibodies may be prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

Various forms of the humanized antibody are contemplated. For example,the humanized antibody may be an antibody fragment, such as a Fabfragments, F(ab′)₂ fragments, Fv fragments, diabodies, triabodies,tetrabodies, single chain antibodies, scFv, scFv-Fc and the like.Alternatively, the humanized antibody may be an intact antibody, such asan intact IgG1 antibody.

(iv) Human Antibodies

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA, 90:2551;Jakobovits et al., 1993, Nature 362:255-258; Bruggermann et al., 1993,Year in Immuno. 7:33; and U.S. Pat. Nos. 5,591,669, 5,589,369 and5,545,807.

Alternatively, phage display technology (see, e.g., McCafferty et al.,1990, Nature 348:552-553) can be used to produce human antibodies andantibody fragments in vitro, from immunoglobulin variable (V) domaingene repertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson and Chiswell,1993, Current Opinion in Structural Biology 3:564-571. Several sourcesof V-gene segments can be used for phage display. Clackson et al., 1991,Nature 352:624-628 isolated a diverse array of anti-oxazolone antibodiesfrom a small random combinatorial library of V genes derived from thespleens of immunized mice. A repertoire of V genes from unimmunizedhuman donors can be constructed and antibodies to a diverse array ofantigens (including self-antigens) can be isolated essentially followingthe techniques described by Marks et al., 1991, J. Mol. Biol.222:581-597), or Griffith et al., 1993, EMBO J. 12:725-734. See alsoU.S. Pat. Nos. 5,565,332 and 5,573,905. As discussed above, humanantibodies may also be generated by in vitro activated B cells (see,e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275). Human anti-CD30antibodies are described in U.S. Patent Application Publication No.2004-0006215.

(v) Antibody Fragments

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., 1992,Journal of Biochemical and Biophysical Methods 24:107-117; and Brennanet al., 1985, Science 229:81). However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from the antibody phage libraries discussedabove. Alternatively, Fab′-SH fragments can be directly recovered fromE. coli and chemically coupled to form F(ab′)₂ fragments (see, e.g.,Carter et al., 1992, Bio/Technology 10: 163-167). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Other techniques for the production of antibodyfragments will be apparent to the skilled practitioner. In otherembodiments, the antibody of choice is a single chain Fv fragment(scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No.5,587,458. The antibody fragment may also be a “linear antibody”, e.g.,as described in U.S. Pat. No. 5,641,870 for example. Such linearantibody fragments may be monospecific or bispecific.

(vi) Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of a target protein. Alternatively, anantibody arm may be combined with an arm which binds to a Fc receptorsfor IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16)so as to focus cellular defense mechanisms to the target-expressingcell. Bispecific antibodies may also be used to localize cytotoxicagents to cells which express the target.

Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (see, e.g., Millsteinet al., 1983, Nature 305:537-539). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., 1991,EMBO J. 10:3655-3659. According to a different approach, antibodyvariable domains with the desired binding specificities(antibody-antigen combining sites) are fused to immunoglobulin constantdomain sequences. The fusion preferably is with an immunoglobulin heavychain constant domain, comprising at least part of the hinge, C_(H)2,and C_(H)3 regions. It is preferred to have the first heavy-chainconstant region (C_(H)1) containing the site necessary for light chainbinding, present in at least one of the fusions. DNAs encoding theimmunoglobulin heavy chain fusions and, if desired, the immunoglobulinlight chain, are inserted into separate expression vectors, and areco-transfected into a suitable host organism. This provides for greatflexibility in adjusting the mutual proportions of the three polypeptidefragments in embodiments when unequal ratios of the three polypeptidechains used in the construction provide the optimum yields. It is,however, possible to insert the coding sequences for two or all threepolypeptide chains in one expression vector when the expression of atleast two polypeptide chains in equal ratios results in high yields orwhen the ratios are of no particular significance.

In one embodiment of this approach, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., 1986, Methods in Enzymology 121:210.

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H)3 domain of an antibody constant domain. In thismethod, one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g.,tyrosine or tryptophan). Compensatory “cavities” of identical or similarsize to the large side chain(s) are created on the interface of thesecond antibody molecule by replacing large amino acid side chains withsmaller ones (e.g., alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al., 1985,Science, 229: 81 describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., 1992, J. Exp. Med. 175: 217-225 describe theproduction of a fully humanized bispecific antibody F(ab′)₂ molecule.Each Fab′ fragment was separately secreted from E. coli and subjected todirected chemical coupling in vitro to form the bispecific antibody.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., 1992, J. Immunol. 148(5):1547-1553.The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., 1993, Proc. Natl.Acad. Sci. USA 90:6444-6448 has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., 1994, J. Immunol. 152:5368.

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

(vii) Other Amino Acid Sequence Modifications

Amino acid sequence modification(s) of the antibodies described hereinare contemplated. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the antibody.Amino acid sequence variants of the antibodies are prepared byintroducing appropriate nucleotide changes into the antibody nucleicacid, or by peptide synthesis. Such modifications include, for example,deletions from, and/or insertions into and/or substitutions of, residueswithin the amino acid sequences of the antibody. Any combination ofdeletion, insertion, and substitution is made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics. The amino acid changes also may alterpost-translational processes of the antibody, such as changing thenumber or position of glycosylation sites.

A useful method for identification of certain residues or regions of theantibody that are favored locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells, 1989,Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed antibodyvariants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g., for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated.

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain.Naturally-occurring residues can be divided into groups based on commonside-chain properties:

-   -   (1) hydrophobic: norleucine, met, ala, val, leu, ile;    -   (2) neutral hydrophilic: cys, ser, thr;    -   (3) acidic: asp, glu;    -   (4) basic: asn, gln, his, lys, arg;    -   (5) residues that influence chain orientation: gly, pro; and    -   (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g., a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g., 6-7 sites) are mutated togenerate all possible amino substitutions at each site. The antibodyvariants thus generated are displayed in a monovalent fashion fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g., binding affinity) asherein disclosed. In order to identify candidate hypervariable regionsites for modification, alanine scanning mutagenesis can be performed toidentify hypervariable region residues contributing significantly toantigen binding. Alternatively, or additionally, it may be beneficial toanalyze a crystal structure of the antigen-antibody complex to identifycontact points between the antibody and the antigen. Such contactresidues and neighboring residues are candidates for substitutionaccording to the techniques elaborated herein. Once such variants aregenerated, the panel of variants is subjected to screening as describedherein and antibodies with superior properties in one or more relevantassays may be selected for further development.

It may be desirable to modify the antibody of the invention with respectto effector function, e.g., so as to enhance antigen-dependentcell-mediated cyotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., 1992, J. Exp Med. 176:1191-1195 and Shopes, 1992, J. Immunol.148:2918-2922. Homodimeric antibodies with enhanced anti-tumor activitymay also be prepared using heterobifunctional cross-linkers as describedin Wolff et al., 1993, Cancer Research 53:2560-2565. Alternatively, anantibody can be engineered which has dual Fc regions and may therebyhave enhanced complement lysis and ADCC capabilities. See Stevenson etal., 1989, Anti-Cancer Drug Design 3:219-230.

To increase the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

(viii) Glycosylation Variants

Antibodies in the ADC of the invention may be glycosylated at conservedpositions in their constant regions (see, e.g., Jefferis and Lund, 1997,Chem. Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32).The oligosaccharide side chains of the immunoglobulins affect theprotein's function (see, e.g., Boyd et al., 1996, Mol. Immunol.32:1311-1318; Wittwe and Howard, 1990, Biochem. 29:4175-4180), and theintramolecular interaction between portions of the glycoprotein whichcan affect the conformation and presented three-dimensional surface ofthe glycoprotein (Hefferis and Lund, supra; Wyss and Wagner, 1996,Current Opin. Biotech. 7:409-416). Oligosaccharides may also serve totarget a given glycoprotein to certain molecules based upon specificrecognition structures. For example, it has been reported that inagalactosylated IgG, the oligosaccharide moiety ‘flips’ out of theinter-C_(H)2 space and terminal N-acetylglucosamine residues becomeavailable to bind mannose binding protein (Malhotra et al., 1995, NatureMed. 1:237-243). Removal by glycopeptidase of the oligosaccharides fromCAMPATH-1H (a recombinant humanized murine monoclonal IgG1 antibodywhich recognizes the CDw52 antigen of human lymphocytes) produced inChinese Hamster Ovary (CHO) cells resulted in a complete reduction incomplement mediated lysis (CMCL) (Boyd et al., 1996, Mol. Immunol.32:1311-1318), while selective removal of sialic acid residues usingneuraminidase resulted in no loss of DMCL. Glycosylation of antibodieshas also been reported to affect antibody-dependent cellularcytotoxicity (ADCC). In particular, CHO cells withtetracycline-regulated expression ofβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing formation of bisecting GlcNAc, wasreported to have improved ADCC activity (Umana et al., 1999, NatureBiotech. 17:176-180).

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Glycosylation variants of antibodies are variants in which theglycosylation pattern of an antibody is altered. By altering is meantdeleting one or more carbohydrate moieties found in the antibody, addingone or more carbohydrate moieties to the antibody, changing thecomposition of glycosylation (glycosylation pattern), the extent ofglycosylation, etc.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).Similarly, removal of glycosylation sites can be accomplished by aminoacid alteration within the native glycosylation sites of the antibody.

The amino acid sequence is usually altered by altering the underlyingnucleic acid sequence. These methods include, but are not limited to,isolation from a natural source (in the case of naturally-occurringamino acid sequence variants) or preparation by oligonucleotide-mediated(or site-directed) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared variant or a non-variant version ofthe antibody.

The glycosylation (including glycosylation pattern) of antibodies mayalso be altered without altering the amino acid sequence or theunderlying nucleotide sequence. Glycosylation largely depends on thehost cell used to express the antibody. Since the cell type used forexpression of recombinant glycoproteins, e.g., antibodies, as potentialtherapeutics is rarely the native cell, significant variations in theglycosylation pattern of the antibodies can be expected. See, e.g., Hseet al., 1997, J. Biol. Chem. 272:9062-9070. In addition to the choice ofhost cells, factors which affect glycosylation during recombinantproduction of antibodies include growth mode, media formulation, culturedensity, oxygenation, pH, purification schemes and the like. Variousmethods have been proposed to alter the glycosylation pattern achievedin a particular host organism including introducing or overexpressingcertain enzymes involved in oligosaccharide production (U.S. Pat. Nos.5,047,335; 5,510,261; and 5,278,299). Glycosylation, or certain types ofglycosylation, can be enzymatically removed from the glycoprotein, forexample using endoglycosidase H (Endo H). In addition, the recombinanthost cell can be genetically engineered, e.g., make defective inprocessing certain types of polysaccharides. These and similartechniques are well known in the art.

The glycosylation structure of antibodies can be readily analyzed byconventional techniques of carbohydrate analysis, including lectinchromatography, NMR, Mass spectrometry, HPLC, GPC, monosaccharidecompositional analysis, sequential enzymatic digestion, and HPAEC-PAD,which uses high pH anion exchange chromatography to separateoligosaccharides based on charge. Methods for releasing oligosaccharidesfor analytical purposes are also known, and include, without limitation,enzymatic treatment (commonly performed using peptide-N-glycosidaseF/endo-β-galactosidase), elimination using harsh alkaline environment torelease mainly O-linked structures, and chemical methods using anhydroushydrazine to release both N- and O-linked oligosaccharides.

Screening for Ligand-Linker-Drug Conjugates

Transgenic animals and cell lines are particularly useful in screeningDrug-Linker-Ligand conjugates (e.g., antibody drug conjugates (ADC)) forprophylactic or therapeutic treatments of diseases or disordersinvolving overexpression of a target protein (e.g., CD20, CD30, CD33,CD40, CD70, BCMA, and Lewis Y). The screening of Drug-Linker-Ligandconjugates as ADCs is exemplified herein;

Transgenic animals and cell lines are particularly useful in screeningantibody drug conjugates (ADC). Screening for a useful ADC may involveadministering candidate ADC over a range of doses to the transgenicanimal, and assaying at various time points for the effect(s) of the ADCon the disease or disorder being evaluated. Alternatively, oradditionally, the drug can be administered prior to or simultaneouslywith exposure to an inducer of the disease, if applicable. Candidate ADCmay be screened serially and individually, or in parallel under mediumor high-throughput screening format. The rate at which ADC may bescreened for utility for prophylactic or therapeutic treatments ofdiseases or disorders is limited only by the rate of synthesis orscreening methodology, including detecting/measuring/analysis of data.

One embodiment is a screening method comprising (a) transplanting cellsfrom a stable renal cell cancer cell line into a non-human animal, (b)administering an ADC drug candidate to the non-human animal and (c)determining the ability of the candidate to inhibit the formation oftumors from the transplanted cell line.

Another embodiment is a screening method comprising (a) contacting cellsfrom a stable Hodgkin's disease cell line with an ADC drug candidate and(b) evaluating the ability of the ADC candidate to block ligandactivation of CD40.

Another embodiment is a screening method comprising (a) contacting cellsfrom a stable Hodgkin's disease cell line with an ADC drug candidate and(b) evaluating the ability of the ADC candidate to induce cell death. Inone embodiment the ability of the ADC candidate to induce apoptosis isevaluated.

One embodiment is a screening method comprising (a) transplanting cellsfrom a stable cancer cell line into a non-human animal, (b)administering an ADC drug candidate to the non-human animal and (c)determining the ability of the candidate to inhibit the formation oftumors from the transplanted cell line.

Another embodiment is a screening method comprising (a) contacting cellsfrom a stable cancer cell line with an ADC drug candidate and (b)evaluating the ability of the ADC candidate to induce cell death. In oneembodiment the ability of the ADC candidate to induce apoptosis isevaluated.

In one embodiment, candidate ADC are screened by being administered tothe transgenic animal over a range of doses, and evaluating the animal'sphysiological response to the compounds over time. Administration may beoral, or by suitable injection, depending on the chemical nature of thecompound being evaluated. In some cases, it may be appropriate toadminister the compound in conjunction with co-factors that wouldenhance the efficacy of the compound. If cell lines derived from thesubject transgenic animals are used to screen for compounds useful intreating various disorders, the test compounds are added to the cellculture medium at an appropriate time, and the cellular response to thecompound is evaluated over time using the appropriate biochemical and/orhistological assays. In some cases, it may be appropriate to apply thecompound of interest to the culture medium in conjunction withco-factors that would enhance the efficacy of the compound.

Thus, provided herein are assays for identifying Drug-Linker-Ligandconjugates (such as ADCs) which specifically target and bind a targetprotein, the presence of which is correlated with abnormal cellularfunction, and in the pathogenesis of cellular proliferation and/ordifferentiation that is causally related to the development of tumors.

To identify growth inhibitory compounds that specifically target anantigen of interest, one may screen for compounds which inhibit thegrowth of cancer cells overexpressing antigen of interest derived fromtransgenic animals, the assay described in U.S. Pat. No. 5,677,171 canbe performed. According to this assay, cancer cells overexpressing theantigen of interest are grown in a 1:1 mixture of F12 and DMEM mediumsupplemented with 10% fetal bovine serum, glutamine and penicillinstreptomycin. The cells are plated at 20,000 cells in a 35 mm cellculture dish (2 mls/35 mm dish) and the test compound is added atvarious concentrations. After six days, the number of cells, compared tountreated cells is counted using an electronic COULTER™ cell counter.Those compounds which inhibit cell growth by about 20-100% or about50-100% may be selected as growth inhibitory compounds.

To select for compounds which induce cell death, loss of membraneintegrity as indicated by, e.g., PI, trypan blue or 7AAD uptake may beassessed relative to control. The PI uptake assay uses cells isolatedfrom the tumor tissue of interest of a transgenic animal. According tothis assay, the cells are cultured in Dulbecco's Modified Eagle Medium(D-MEM):Ham's F-12 (50:50) supplemented with 10% heat-inactivated FBS(Hyclone) and 2 mM L-glutamine. Thus, the assay is performed in theabsence of complement and immune effector cells. The cells are seeded ata density of 3×10⁶ per dish in 100×20 mm dishes and allowed to attachovernight. The medium is then removed and replaced with fresh mediumalone or medium containing various concentrations of the compound. Thecells are incubated for a 3-day time period. Following each treatment,monolayers are washed with PBS and detached by trypsinization. Cells arethen centrifuged at 1200 rpm for 5 minutes at 4° C., the pelletresuspended in 3 ml cold Ca²⁺ binding buffer (10 mM Hepes, pH 7.4, 140mM NaCl, 2.5 mM CaCl₂) and aliquoted into 35 mm strainer-capped 12×75 mmtubes (1 ml per tube, 3 tubes per treatment group) for removal of cellclumps. Tubes then receive PI (10 μg/ml). Samples may be analyzed usinga FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software (BectonDickinson). Those compounds which induce statistically significantlevels of cell death as determined by PI uptake may be selected as celldeath-inducing compounds.

In order to select for compounds which induce apoptosis, an annexinbinding assay using cells established from the tumor tissue of interestof the transgenic animal is performed. The cells are cultured and seededin dishes as discussed in the preceding paragraph. The medium is thenremoved and replaced with fresh medium alone or medium containing 10μg/ml of the antibody drug conjugate (ADC). Following a three-dayincubation period, monolayers are washed with PBS and detached bytrypsinization. Cells are then centrifuged, resuspended in Ca²⁺ bindingbuffer and aliquoted into tubes as discussed above for the cell deathassay. Tubes then receive labeled annexin (e.g., annexin V-FITC) (1μg/ml). Samples may be analyzed using a FACSCAN™ flow cytometer andFACSCONVERT™ CellQuest software (Becton Dickinson). Those compoundswhich induce statistically significant levels of annexin bindingrelative to control are selected as apoptosis-inducing compounds.

In Vitro Cell Proliferation Assays

Generally, the cytotoxic or cytostatic activity of a Drug-Linker-Ligandconjugate, such as an antibody drug conjugate (ADC), is measured by:exposing mammalian cells having receptor proteins to the antibody of theconjugate in a cell culture medium; culturing the cells for a periodfrom about 6 hours to about 5 days; and measuring cell viability.Cell-based in vitro assays are used to measure viability(proliferation), cytotoxicity, and induction of apoptosis (caspaseactivation) of a Drug-Linker-Ligand conjugate. The screening ofDrug-Linker-Ligand conjugates as ADCs is exemplified herein.

The in vitro potency of antibody drug conjugates is measured by a cellproliferation assay (see Examples). The CellTiter-Glo® Luminescent CellViability Assay is a commercially available (Promega Corp., Madison,Wis.), homogeneous assay method based on the recombinant expression ofColeoptera luciferase (U.S. Pat. Nos. 5,583,024; 5,674,713 and5,700,670). This cell proliferation assay determines the number ofviable cells in culture based on quantitation of the ATP present, anindicator of metabolically active cells (Crouch et al., 1993, J.Immunol. Meth. 160:81-88, U.S. Pat. No. 6,602,677). The CellTiter-Glo®Assay is conducted in 96 well format, making it amenable to automatedhigh-throughput screening (HTS) (Cree et al. (1995) AntiCancer Drugs6:398-404). The homogeneous assay procedure involves adding the singlereagent (CellTiter-Glo® Reagent) directly to cells cultured inserum-supplemented medium. Cell washing, removal of medium and multiplepipetting steps are not required. The system detects as few as 15cells/well in a 384-well format in 10 minutes after adding reagent andmixing. The cells may be treated continuously with ADC, or they may betreated and separated from ADC. Generally, cells treated briefly, i.e.,3 hours, show the same potency effects as continuously treated cells.

The homogeneous “add-mix-measure” format results in cell lysis andgeneration of a luminescent signal proportional to the amount of ATPpresent. The amount of ATP is directly proportional to the number ofcells present in culture. The CellTiter-Glo® Assay generates a“glow-type” luminescent signal, produced by the luciferase reaction,which has a half-life generally greater than five hours, depending oncell type and medium used. Viable cells are reflected in relativeluminescence units (RLU). The substrate, Beetle Luciferin, isoxidatively decarboxylated by recombinant firefly luciferase withconcomitant conversion of ATP to AMP and generation of photons. Theextended half-life eliminates the need to use reagent injectors andprovides flexibility for continuous or batch mode processing of multipleplates. This cell proliferation assay can be used with various multiwellformats, e.g., 96 or 384 well format. Data can be recorded byluminometer or CCD camera imaging device. The luminescence output ispresented as relative light units (RLU), measured over time.

The anti-proliferative effects of antibody drug conjugates can bemeasured by the cell proliferation, in vitro cell killing assay aboveagainst different breast tumor cell lines.

In Vivo Plasma Clearance and Stability

Pharmacokinetic plasma clearance and stability of Drug-Linker-Ligandconjugates, such as ADCs, can be investigated in rats and cynomolgusmonkeys over time. The screening of Drug-Linker-Ligand conjugates asADCs is exemplified herein.

Rodent Toxicity

Antibody drug conjugates and an ADC-minus control, “Vehicle”, areevaluated in an acute toxicity rat model. Toxicity of ADC isinvestigated by treatment of male and female Sprague-Dawley rats withthe ADC and subsequent inspection and analysis of the effects on variousorgans. Gross observations include changes in body weights and signs oflesions and bleeding. Clinical pathology parameters (serum chemistry andhematology), histopathology, and necropsy are conducted on dosedanimals. It is considered that weight loss, or weight change relative toanimals dosed only with Vehicle, in animals after dosing with ADC is agross and general indicator of systemic or localized toxicity.

Hepatotoxicity is measured by elevated liver enzymes, increased numbersof mitotic and apoptotic figures and hepatocyte necrosis. Hematolymphoidtoxicity is observed by depletion of leukocytes, primarily granuloctyes(neutrophils), and/or platelets, and lymphoid organ involvement, i.e.atrophy or apoptotic activity. Toxicity is also noted bygastrointestinal tract lesions such as increased numbers of mitotic andapoptotic figures and degenerative enterocolitis.

Enzymes indicative of liver injury that are studied include:

AST (aspartate aminotransferase)

Localization: cytoplasmic; liver, heart, skeletal muscle, kidney

Liver:Plasma ratio of 7000:1

T1/2: 17 hrs

ALT (alanine aminotransferase)

Localization: cytoplasmic; liver, kidney, heart, skeletal muscle

Liver:Plasma ratio of 3000:1

-   -   T1/2: 42 hrs; diurnal variation    -   GGT (g-glutamyl transferase)    -   Localization: plasma membrane of cells with high secretory or        absorptive capacity; liver, kidney, intestine        Poor predictor of liver injury; commonly elevated in bile duct        disorders

Cynomolgus Monkey Toxicity/Safety

Similar to the rat toxicity/safety study, cynomolgus monkeys are treatedwith ADC followed by liver enzyme measurements, and inspection andanalysis of the effects on various organs. Gross observations includechanges in body weights and signs of lesions and bleeding. Clinicalpathology parameters (serum chemistry and hematology), histopathology,and necropsy are conducted on dosed animals.

Synthesis of the Compounds

The Exemplary Compounds and Exemplary Conjugates can be made using thesynthetic procedures outlined below in Schemes 5-16. As described inmore detail below, the Exemplary Compounds or Exemplary Conjugates canbe conveniently prepared using a Linker having a reactive site forbinding to the Drug and Ligand. In one aspect, a Linker has a reactivesite which has an electrophilic group that is reactive to a nucleophilicgroup present on a Ligand, such as but not limited to an antibody.Useful nucleophilic groups on an antibody include but are not limitedto, sulfhydryl, hydroxyl and amino groups. The heteroatom of thenucleophilic group of an antibody is reactive to an electrophilic groupon a Linker and forms a covalent bond to a Linker unit. Usefulelectrophilic groups include, but are not limited to, maleimide andhaloacetamide groups. The electrophilic group provides a convenient sitefor antibody attachment.

In another embodiment, a Linker has a reactive site which has anucleophilic group that is reactive to an electrophilic group present onan antibody. Useful electrophilic groups on an antibody include, but arenot limited to, aldehyde and ketone carbonyl groups. The heteroatom of anucleophilic group of a Linker can react with an electrophilic group onan antibody and form a covalent bond to an antibody unit. Usefulnucleophilic groups on a Linker include, but are not limited to,hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazinecarboxylate, and arylhydrazide. The electrophilic group on an antibodyprovides a convenient site for attachment to a Linker.

Carboxylic acid functional groups and chloroformate functional groupsare also useful reactive sites for a Linker because they can react withsecondary amino groups of a Drug to form an amide linkage. Also usefulas a reactive site is a carbonate functional group on a Linker, such asbut not limited to p-nitrophenyl carbonate, which can react with anamino group of a Drug, such as but not limited to N-methyl valine, toform a carbamate linkage. Typically, peptide-based Drugs can be preparedby forming a peptide bond between two or more amino acids and/or peptidefragments. Such peptide bonds can be prepared, for example, according tothe liquid phase synthesis method (see Schröder and Lübke, “ThePeptides”, volume 1, pp 76-136, 1965, Academic Press) that is well knownin the field of peptide chemistry.

The synthesis of an illustrative Stretcher having an electrophilicmaleimide group is illustrated below in Schemes 8-9. General syntheticmethods useful for the synthesis of a Linker are described in Scheme 10.Scheme 11 shows the construction of a Linker unit having a val-citgroup, an electrophilic maleimide group and a PAB self-immolative Spacergroup. Scheme 12 depicts the synthesis of a Linker having a phe-lysgroup, an electrophilic maleimide group, with and without the PABself-immolative Spacer group. Scheme 13 presents a general outline forthe synthesis of a Drug-Linker Compound, while Scheme 14 presents analternate route for preparing a Drug-Linker Compound. Scheme 15 depictsthe synthesis of a branched linker containing a BHMS group. Scheme 16outlines the attachment of an antibody to a Drug-Linker Compound to forma Drug-Linker-Antibody Conjugate, and Scheme 14 illustrates thesynthesis of Drug-Linker-Antibody Conjugates having, for example but notlimited to, 2 or 4 drugs per Antibody.

As described in more detail below, the Exemplary Conjugates areconveniently prepared using a Linker having two or more Reactive Sitesfor binding to the Drug and a Ligand. In one aspect, a Linker has aReactive site which has an electrophilic group that is reactive to anucleophilic group present on a Ligand, such as an antibody. Usefulnucleophilic groups on an antibody include but are not limited to,sulfhydryl, hydroxyl and amino groups. The heteroatom of thenucleophilic group of an antibody is reactive to an electrophilic groupon a Linker and forms a covalent bond to a Linker unit. Usefulelectrophilic groups include, but are not limited to, maleimide andhaloacetamide groups. The electrophilic group provides a convenient sitefor antibody attachment.

In another embodiment, a Linker has a Reactive site which has anucleophilic group that is reactive to an electrophilic group present ona Ligand, such as an antibody. Useful electrophilic groups on anantibody include, but are not limited to, aldehyde and ketone carbonylgroups. The heteroatom of a nucleophilic group of a Linker can reactwith an electrophilic group on an antibody and form a covalent bond toan antibody unit. Useful nucleophilic groups on a Linker include, butare not limited to, hydrazide, oxime, amino, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. Theelectrophilic group on an antibody provides a convenient site forattachment to a Linker.

In yet another embodiment a Drug containing aromatic arsine oxide candirectly bind to a Ligand unit containing proximal dithiols to formstable arsine-dithiol cyclic structures.

Drug Moiety Synthesis

Typically, peptide-based Drugs can be prepared by forming a peptide bondbetween two or more amino acids and/or peptide fragments. Such peptidebonds can be prepared, for example, according to the liquid phasesynthesis method (see E. Schröder and K. Lübke, “The Peptides”, volume1, pp 76-136, 1965, Academic Press) that is well known in the field ofpeptide chemistry.

The auristatin/dolastatin drug moieties may be prepared according to thegeneral methods of: U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588;Pettit et al., 1989, J. Am. Chem. Soc. 111:5463-5465; Pettit et al.,1998, Anti-Cancer Drug Design 13:243-277; and Pettit et al., 1996, J.Chem. Soc. Perkin Trans. 15:859-863.

In one embodiment, a Drug is prepared by combining about astoichiometric equivalent of a dipeptide and a tripeptide, preferably ina one-pot reaction under suitable condensation conditions. This approachis illustrated in Schemes 5-7, below.

Scheme 5 illustrates the synthesis of an N-terminal tripeptide unit Fwhich is a useful intermediate for the synthesis of the drug compoundsof Formula Ib.

As illustrated in Scheme 5, a protected amino acid A (where PGrepresents an amine protecting group, R⁴ is selected from hydrogen,C₁-C₈ alkyl, C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, X¹-aryl,X¹—(C₃-C₈ carbocycle), C₃-C₈ heterocycle, X¹—(C₃-C₈ heterocycle) and R⁵is selected from H and methyl; or R⁴ and R⁵ join, have the formula—(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selectedfrom hydrogen, C₁-C₈ alkyl and C₃-C₈ carbocycle and n is selected from2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they areattached) is coupled to t-butyl ester B (where R⁶ is selected from —Hand —C₁-C₈ alkyl; and R⁷ is selected from hydrogen, C₁-C₈ alkyl, C₃-C₈carbocycle, —O—(C₁-C₈ alkyl), aryl, X¹-aryl, X¹—(C₃-C₈ carbocycle),C₃-C₈ heterocycle and X¹—(C₃-C₈ heterocycle)) under suitable couplingconditions, e.g., in the presence of PyBrop and diisopropylethylamine,or using DCC (see, for example, Miyazaki et. al., 1995, Chem. Pharm.Bull. 43(10):1706-1718).

Suitable protecting groups PG, and suitable synthetic methods to protectan amino group with a protecting group are well known in the art. See,e.g., Greene and Wuts, Protective Groups in Organic Synthesis, 2ndEdition, 1991, John Wiley & Sons. Exemplary protected amino acids A arePG-e and, particularly, PG-Val, while other suitable protected aminoacids include, without limitation: PG-cyclohexylglycine,PG-cyclohexylalanine, PG-aminocyclopropane-1-carboxylic acid,PG-aminoisobutyric acid, PG-phenylalanine, PG-phenylglycine, andPG-tert-butylglycine. Z is an exemplary protecting group. Fmoc isanother exemplary protecting group. An exemplary t-butyl ester B isdolaisoleuine t-butyl ester.

The dipeptide C can be purified, e.g., using chromatography, andsubsequently deprotected, e.g., using H2 and 10% Pd—C in ethanol when PGis benzyloxycarbonyl, or using diethylamine for removal of an Fmocprotecting group. The resulting amine D readily forms a peptide bondwith an amino acid BB (wherein R¹ is selected from —H, —C₁-C₈ alkyl and—C₃-C₈ carbocycle; and R² is selected from —H and —C₁-C₈ alkyl; or R¹and R² join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a) andR^(b) are independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring withthe nitrogen atom to which they are attached; and R³ is selected fromhydrogen, C₁-C₈ alkyl, C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), aryl,X¹-aryl, X¹—(C₃-C₈ carbocycle), C₃-C₈ heterocycle and X¹—(C₃-C₈heterocycle)). N,N-Dialkyl amino acids are exemplary amino acids for BB,such as commercially available N,N-dimethyl valine. Other N,N-dialkylamino acids can be prepared by reductive bis-alkylation using knownprocedures (see, e.g., Bowman et al., 1950, J. Chem. Soc. 1342-1340).Fmoc-Me-L-Val and Fmoc-Me-L-glycine are two exemplary amino acids BBuseful for the synthesis of N-monoalkyl derivatives. The amine D and theamino acid BB react to provide the tripeptide E using coupling reagentDEPC with triethylamine as the base. The C-terminus protecting group ofE is subsequently deprotected using HCl to provide the tripeptidecompound of formula F.

Illustrative DEPC coupling methodology and the PyBrop couplingmethodology shown in Scheme 5 are outlined below in General Procedure Aand General Procedure B, respectively. Illustrative methodology for thedeprotection of a CBZ-protected amine via catalytic hydrogenation isoutlined below in General Procedure C.

General Procedure A: Peptide Synthesis Using DEPC.

The N-protected or N,N-disubstituted amino acid or peptide D (1.0 eq.)and an amine BB (1.1 eq.) are diluted with an aprotic organic solvent,such as dichloromethane (0.1 to 0.5 M). An organic base such astriethylamine or diisopropylethylamine (1.5 eq.) is then added, followedby DEPC (1.1 eq.). The resulting solution is stirred, preferably underargon, for up to 12 hours while being monitored by HPLC or TLC. Thesolvent is removed in vacuo at room temperature, and the crude productis purified using, for example, HPLC or flash column chromatography(silica gel column). Relevant fractions are combined and concentrated invacuo to afford tripeptide E which is dried under vacuum overnight.

General Procedure B: Peptide Synthesis Using PyBrop.

The amino acid B (1.0 eq.), optionally having a carboxyl protectinggroup, is diluted with an aprotic organic solvent such asdichloromethane or DME to provide a solution of a concentration between0.5 and 1.0 mM, then diisopropylethylamine (1.5 eq.) is added. Fmoc- orCBZ-protected amino acid A (1.1 eq.) is added as a solid in one portion,then PyBrop (1.2 eq.) is added to the resulting mixture. The reaction ismonitored by TLC or HPLC, followed by a workup procedure similar to thatdescribed in General Procedure A.

General Procedure C: Z-Removal Via Catalytic Hydrogenation.

CBZ-protected amino acid or peptide C is diluted with ethanol to providea solution of a concentration between 0.5 and 1.0 mM in a suitablevessel, such as a thick-walled round bottom flask. 10% palladium oncarbon is added (5-10% w/w) and the reaction mixture is placed under ahydrogen atmosphere. Reaction progress is monitored using HPLC and isgenerally complete within 1-2 h. The reaction mixture is filteredthrough a pre-washed pad of celite and the celite is again washed with apolar organic solvent, such as methanol after filtration. The eluentsolution is concentrated in vacuo to afford a residue which is dilutedwith an organic solvent, preferably toluene. The organic solvent is thenremoved in vacuo to afford the deprotected amine C.

Scheme 6 shows a method useful for making a C-terminal dipeptide offormula K and a method for coupling the dipeptide of formula K with thetripeptide of formula F to make drug compounds of Formula Ib. Thismethod is applicable for the phenylalanine replacement moieties H havingacid labile carboxyl protecting group, preferably dimethoxybenzyl group.

The dipeptide K can be readily prepared by condensation of theN-protected modified amino acid PG-Dolaproine (G) with an amine offormula H using condensing agents well known for peptide chemistry, suchas, for example, DEPC in the presence of triethylamine, as shown inSchemes 5 and 6. Suitable N-protected groups for Dolaproine include, butare not limited to, an Fmoc-protecting group.

The dipeptide of formula K can then be coupled with a tripeptide offormula F using General Procedure D to make the Fmoc-protected drugcompounds of formula L which can be subsequently deprotected usingGeneral Procedure E in order to provide the drug compounds of formula(Ib).

General Procedure D: Drug Synthesis.

A mixture of dipeptide K (1.0 eq.) and tripeptide F (1 eq.) is dilutedwith an aprotic organic solvent, such as dichloromethane, to form a 0.1Msolution, then a strong acid, such as trifluoroacetic acid (½ v/v) isadded and the resulting mixture is stirred under a nitrogen atmospherefor two hours at 0° C. The reaction can be monitored using TLC or,preferably, HPLC. The solvent is removed in vacuo and the resultingresidue is azeotropically dried twice, preferably using toluene. Theresulting residue is dried under high vacuum for 12 h and then dilutedwith and aprotic organic solvent, such as dichloromethane. An organicbase such as triethylamine or diisopropylethylamine (1.5 eq.) is thenadded, followed by either PyBrop (1.2 eq.) or DEPC (1.2 eq.) dependingon the chemical functionality on the residue. The reaction mixture ismonitored by either TLC or HPLC and upon completion, the reaction issubjected to a workup procedure similar or identical to that describedin General Procedure A.

General Procedure E: Fmoc-Removal Using Diethylamine.

An Fmoc-protected Drug L is diluted with an aprotic organic solvent suchas dichloromethane and to the resulting solution is added diethylamine(½ v/v). Reaction progress is monitored by TLC or HPLC and is typicallycomplete within 2 h. The reaction mixture is concentrated in vacuo andthe resulting residue is azeotropically dried, preferably using toluene,then dried under high vacuum to afford Drug Ib having a deprotectedamino group. Thus, the above method is useful for making Drugs that canbe used in the present invention.

Alternatively Drug Compounds can be conveniently prepared by solid phasepeptide synthesis using standard Fmoc chemistry well known in the art(see, e.g., Novabiochem® catalogue 2006/2007, Synthesis Notes) as shownin Scheme 6a (infra). Fmoc-protected amino acids can be prepared fromunprotected amino acids using, for example, Fmoc-OSu via wellestablished procedures (see, e.g., Greene and Wuts, Protective Groups inOrganic Synthesis, 2nd Edition, 1991, John Wiley & Sons, p. 506).

Amino acids not commercially available pre-loaded on an appropriate acidlabile resin, preferably 2-chlorotrityl resin, can be loaded onto2-chlorotrityl chloride resin as described in General Procedure SP(a).Loading can be determined by spectrophotometric Fmoc-quantitation assay.Loading levels (mmol/g) of commercially available pre-loaded amino acidson chlorotrityl resin can be determined as described in GeneralProcedure SP(b). Peptides can then be assembled on the resin loaded withthe first amino acid by coupling Fmoc-Dolaproine using appropriatecoupling agent, preferably HATU/DIEA, followed by Fmoc deprotection andsubsequent coupling of Fmoc-MeVal-Val-Dil tripeptide. Solid phasecoupling routine is well established in the art and is described inGeneral Procedure SP(c). Final deprotection of peptides and cleavage offresin can be readily performed following General Procedure SP(d).

General Procedure SP(a). Resin Loading

Fmoc-amino acid (0.84 μmmol) is suspended in anhydrous CH₂Cl₂ (4 mL) andDIEA (585 μL, 3.36 mmol, 4 equiv). The resulting mixture is added to a10-mL syringe containing 2-Chlorotryityl chloride resin (500 mg, 0.70mmol, 1.4 mmol/g). Mixture is agitated for 6 hours at room temperature.Then the resin is filtered, washed with DCM/MeOH/DIEA (17:2:1; 4×5 mL),MeOH (1×5 mL), DCM (4×5 mL), DMF (4×5 mL), DCM (2×5 mL), ethyl ether(4×5 mL), and is dried in vacuo for 2 h. The resin is then left undervacuum overnight to produce resin SP1.

Loading is determined by Fmoc-quantitation. A known quantity (4.4 mg)SP1 resin is weighed into a 10-mL volumetric flask. To the flask istransferred 20% piperidine/DMF (2 mL). The mixture is allowed to cleavefor 1 h, with occasional agitation by hand. To flask is transferred DMF(8 mL) to bring the total volume to 10 mL. A blank solution is preparedwith 10 mL, 20% piperidine/DMF in a 10-mL volumetric flask. Thespectrophotometer is zeroed with the blank solution. The absorbance ismeasured at 301 nm and the loading level is given by:Loading (mmol/g)=A ₃₀₁×10 mL/7800×wtwhereby A₃₀₁ is the absorbance at 301 nm, 7800 is the extinctioncoefficient of the piperidine-fluorenone adduct, and wt is the weight ofresin used in milligrams. Fmoc quantitation is generally performed induplicate.

General Procedure SP(b). Fmoc Quantitation of Commercially AvailablePre-Loaded Resins

Fmoc-Cl (259 mg, 1 mmol) is dissolved in anhydrous CH₂Cl₂ (2 mL) to makea 0.5M working solution. This solution is transferred to a 3-mL plasticsyringe containing Aminoacid-2-Chlorotrityl resin (0.86 mmol/g, 0.0215mmol). The mixture is agitated for 2 h. The resin is then filtered andwashed with DMF (2×5 mL), CH₂Cl₂ (2×5 mL), ethyl ether (2×5 mL), anddried in-vacuo for 2 h. The resin is subjected to Kaiser amine test.Upon negative results (free amine fully protected) the Fmoc quantitationto obtain loading level is performed as shown in General ProcedureSP(a).

General Procedure SP(c). Solid Phase Peptide Coupling Using HATU.

A 20% piperidine in DMF solution (5 μL) is added to the syringecontaining SP1 resin, and the mixture is agitated for 2 h. The resin isthen filtered, washed with DMF (4×5 mL), DCM (4×5 mL), DMF (4×5 mL), DCM(4×5 mL), ethyl ether (4×5 μL), and is dried in-vacuo for 2 h.

Fmoc-Dap (278 mg, 0.680 mmol) and HATU (259 mg, 0.680 mmol, 2 equiv.)are suspended in anhydrous DMF (5 mL) and DIEA (237 μL, 1.36 mmol, 4equiv.). The resulting mixture is transferred to the 10-mL plasticsyringe containing H-Aminoacid-2-Chlorotrityl Resin (555.6 mg, 0.340mmol). The mixture is agitated overnight, at room temperature. Reactioncompletion is determined by Kaiser amine test and LCMS analysis ofmaterial cleaved off a small amount of resin (using 2% TFA/CH₂Cl₂). Theresin is then filtered, washed with DMF (4×5 mL), DCM (4×5 mL), DMF (4×5mL), DCM (4×5 mL), ethyl ether (4×5 mL), and is dried in vacuo for 2 h.

A 20% piperidine in DMF solution (5 mL) is added to the syringecontaining Fmoc-Dap-Aminoacid-2-Chlorotrityl Resin, and the mixture isagitated for 2 h. The resin is filtered, washed with DMF (4×5 mL), DCM(4×5 mL), DMF (4×5 mL), DCM (4×5 mL), ethyl ether (4×5 mL), and is driedin vacuo for 2 h.

Fmoc-MeVal-Val-Dil-OH (510 mg, 0.680 mmol, 2 equiv.) and HATU (259 mg,0.680 mmol, 2 equiv.) are suspended in anhydrous DMF (5-mL) and DIEA(237 μL, 1.70 mmol, 5 equiv.). The resulting mixture is transferred tothe 10-mL plastic syringe containing H-Dap-Aminoacid-2-Chlorotritylresin. The mixture is agitated for 6 h. Reaction completion isdetermined by LCMS analysis of material cleaved off a small amount ofresin (using 2% TFA/CH₂Cl₂). The resin is then filtered, washed with DMF(4×5 mL), DCM (4×5 mL), DMF (4×5 mL), DCM (4×5 mL), ethyl ether (4×5mL), and is dried in vacuo for 2 h.

General Procedure SP(d). Final Deprotection and Cleavage Off Resin.

A 20% piperidine in DMF solution (5 mL) is added to the syringecontaining Fmoc-MeVal-Val-Dil-Dap-Aminoacid-2-Chlorotrityl resin, andthe mixture is agitated for 2 h. The resin is filtered, washed with DMF(4×5 mL), DCM (4×5 mL), DMF (4×5 mL), DCM (4×5 mL), ethyl ether (4×5mL), and is dried in vacuo for 2 h. Further drying can be achieved ifnecessary by leaving resin overnight under vacuum.

A 2% TFA/CH₂Cl₂ (5 mL) solution is transferred to a 10-mL plasticsyringe containing MeVal-Val-Dil-Dap-Aminoacid-2-Chlorotrityl resin andmixture is agitated, at room temperature, for 5 minutes. Filtrate iscollected in a 100 mL round-bottom flask. The process is repeated threetimes. Filtrate is evaporated to leave white solid. Peptides Ib can beisolated by preparative HPLC.

Drug-Linker Synthesis

To prepare a Drug-Linker Compound of the present invention, the Drug isreacted with a reactive site on the Linker. In general, the Linker canhave the structure:

when both a Spacer unit (—Y—) and a Stretcher unit (-A-) are present.Alternately, the Linker can have the structure:

when the Spacer unit (—Y—) is absent.

The Linker can also have the structure:

when both the Stretcher unit (-A-) and the Spacer unit (—Y—) are absent.

The Linker can also have the structure:

when both the Amino Acid unit (W) and the Spacer Unit (Y) are absent.

In general, a suitable Linker has an Amino Acid unit linked to anoptional Stretcher Unit and an optional Spacer Unit. Reactive Site 1 ispresent at the terminus of the Spacer and Reactive site 2 is present atthe terminus of the Stretcher. If a Spacer unit is not present, thenReactive site 1 is present at the C-terminus of the Amino Acid unit.

In an exemplary embodiment, Reactive Site No. 1 is reactive to anitrogen atom of the Drug, and Reactive Site No. 2 is reactive to asulfhydryl group on the Ligand. Reactive Sites 1 and 2 can be reactiveto different functional groups.

In another exemplary embodiment, Reactive Site No. 2 is reactive toamino groups on lysines on the Ligand.

In one aspect of the invention, Reactive Site No. 1 is

In another aspect, Reactive Site No. 1 is

In still another aspect, Reactive Site No. 1 is a p-nitrophenylcarbonate having the formula

In one aspect of, Reactive Site No. 2 is a thiol-accepting group.Suitable thiol-accepting groups include haloacetamide groups having theformula

wherein X represents a leaving group, preferably O-mesyl, O-tosyl, —Cl,—Br, or —I; or a maleimide group having the formula

Useful Linkers can be obtained via commercial sources, such as MolecularBiosciences Inc. (Boulder, Colo.), or prepared as summarized in Schemes8-10 below.

wherein X is —CH₂— or —CH₂OCH₂—; and n is an integer ranging either from0-10 when X is —CH₂—; or 1-10 when X is —CH₂OCH₂—.

The method shown in Scheme 9 combines maleimide with a glycol underMitsunobu conditions to make a polyethylene glycol maleimide Stretcher(see, e.g., example, Walker, 1995, J. Org. Chem. 60, 5352-5), followedby installation of a p-nitrophenyl carbonate Reactive Site group.

wherein E is —CH₂— or —CH₂OCH₂—; and e is an integer ranging from 0-8;

Alternatively, PEG-maleimide and PEG-haloacetamide stretchers can beprepared as described by Frisch et al., 1996, Bioconjugate Chem.7:180-186.

Scheme 10 illustrates a general synthesis of an illustrative Linker unitcontaining a maleimide Stretcher group and optionally a p-aminobenzylether self-immolative Spacer.

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;m is an integer ranging from 0-4; and n is an integer ranging from 0-10.

Useful Stretchers may be incorporated into a Linker using thecommercially available intermediates from Molecular Biosciences(Boulder, Colo.) described below by utilizing known techniques oforganic synthesis.

Stretchers of formula (IIIa) can be introduced into a Linker by reactingthe following intermediates with the N-terminus of an Amino Acid unit asdepicted in Schemes 11 and 12:

-   -   where n is an integer ranging from 1-10 and T is —H or —SO₃Na;

-   -   where n is an integer ranging from 0-3;

Stretcher units of formula (IIIb) can be introduced into a Linker byreacting the following intermediates with the N-terminus of an AminoAcid unit:

Stretcher units of formula (IV) can be introduced into a Linker byreacting the following intermediates with the N-terminus of an AminoAcid unit:

Stretcher units of formula (Va) can be introduced into a Linker byreacting the following intermediates with the N-terminus of an AminoAcid unit:

Other useful Stretchers may be synthesized according to knownprocedures. Aminooxy Stretchers of the formula shown below can beprepared by treating alkyl halides with N-Boc-hydroxylamine according toprocedures described in Jones et al., 2000, Tetrahedron Letters41(10):1531-1533; and Gilon et al., 1967, Tetrahedron 23(11):4441-4447.

wherein —R¹⁷— is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-,—O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-, -arylene-C₁-C₁₀alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-, —(C₃-C₈carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈ heterocyclo-, —C₁-C₁₀alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈ heterocyclo)-C₁-C₁₀ alkylene-,—(CH₂CH₂O)_(r)—, —(CH₂CH₂O)_(r)—CH₂—; and r is an integer ranging from1-10;

Isothiocyanate Stretchers of the formula shown below may be preparedfrom isothiocyanatocarboxylic acid chlorides as described in Angew.Chem., 87(14):517 (1975).

-   -   wherein —R¹⁷— is as described herein.

Scheme 11 shows a method for obtaining of a val-cit dipeptide Linkerhaving a maleimide Stretcher and optionally a p-aminobenzylself-immolative Spacer.

-   -   wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or        -cyano; and m is an integer ranging from 0-4.

Scheme 12 illustrates the synthesis of a phe-lys(Mtr) dipeptide Linkerunit having a maleimide Stretcher unit and a p-aminobenzylself-immolative Spacer unit. Starting material AD (lys(Mtr)) iscommercially available (Bachem, Torrance, Calif.) or can be preparedaccording to Dubowchik, et al., 1997 Tetrahedron Letters 38:5257-60.

-   -   wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or        -cyano; and m is an integer ranging from 0-4.

As shown in Scheme 13, a Linker can be reacted with an amino group of aDrug Compound of Formula (Ib) to form a Drug-Linker Compound thatcontains an amide or carbamate group, linking the Drug unit to theLinker unit. When Reactive Site No. 1 is a carboxylic acid group, as inLinker AJ, the coupling reaction can be performed using HATU or PyBropand an appropriate amine base, resulting in a Drug-Linker Compound AK,containing an amide bond between the Drug unit and the Linker unit. WhenReactive Site No. 1 is a carbonate, as in Linker AL, the Linker can becoupled to the Drug using HOBt in a mixture of DMF/pyridine to provide aDrug-Linker Compound AM, containing a carbamate bond between the Drugunit and the Linker unit.

Alternately, when Reactive Site No. 1 is a good leaving group, such asin Linker AN, the Linker can be coupled with an amine group of a Drugvia a nucleophilic substitution process to provide a Drug-LinkerCompound having an amine linkage (AO) between the Drug unit and theLinker unit.

Illustrative methods useful for linking a Drug to a Ligand to form aDrug-Linker Compound are depicted in Scheme 13 and are outlined inGeneral Procedures G-H.

General Procedure G: Amide Formation Using HATU.

A Drug (Ib) (1.0 eq.) and an N-protected Linker containing a carboxylicacid Reactive site (1.0 eq.) are diluted with a suitable organicsolvent, such as dichloromethane, and the resulting solution is treatedwith HATU (1.5 eq.) and an organic base, preferably pyridine (1.5 eq.).The reaction mixture is allowed to stir under an inert atmosphere,preferably argon, for 6 hours, during which time the reaction mixture ismonitored using HPLC. The reaction mixture is concentrated and theresulting residue is purified using HPLC to yield the amide of formulaAK.

Procedure H: Carbamate Formation Using HOBt.

A mixture of a Linker AL having a p-nitrophenyl carbonate Reactive site(1.1 eq.) and Drug (Ib) (1.0 eq.) are diluted with an aprotic organicsolvent, such as DMF, to provide a solution having a concentration of50-100 mM, and the resulting solution is treated with HOBt (0.2 eq.) andplaced under an inert atmosphere, preferably argon. The reaction mixtureis allowed to stir for 15 min, then an organic base, such as pyridine (¼v/v), is added and the reaction progress is monitored using HPLC. TheLinker is typically consumed within 16 h. The reaction mixture is thenconcentrated in vacuo and the resulting residue is purified using, forexample, HPLC to yield the carbamate AM.

General Procedure S: Amide bond formation between the Linker and theDrug

A Linker containing carboxylic acid (30 mg), and anhydrous DMF (10 μl)are placed under an inert atmosphere, preferably argon, and cooled ondry ice for about 5 min. To this mixture oxalyl chloride (1 mL) wasadded dropwise by syringe. Typically, after few minutes, the mixture isallowed to warm up to room temperature and left for 30 min withoccasional manual stirring. Volatiles are removed under reducedpressure. The residue is re-suspended in anhydrous CH₂Cl₂ (1 mL) and thesolvent is removed in vacuo. The residue is dried at vacuum pumpovernight to produce Linker AN.

The acylchloride AN is suspended in anhydrous CH₂Cl₂ (3 mL). A Drug Ib(0.006 mmol) and N,N-diisopropylethylamine (4 μl, ˜4 eq.) are suspendedin anhydrous CH₂Cl₂ (100 μl) and the mixtures is cooled on the ice bathtypically for about 10 min. To this mixture, 150 μl of the acylchloridein CH₂Cl₂ (˜1.1 eq.) are added via syringe. After 15 min on ice,reaction mixture is allowed to warm up to room temperature and stirringcontinued for about 2 more hours. Reaction progress can be monitored byRP-HPLC. Solvent then is removed in vacuo. The residue is suspended inDMSO (0.5 mL). Water (100 μl) was added and after 0.5 h the mixture ispurified, for example, using preparative HPLC to yield Drug-Linker AO.

General Procedure T: N-Hydroxysuccinimide Ester Linker-Drug Preparation

Scheme 13a depictures example of preparation Linker-Drug Compounds AA2containing N-hydroxysuccinimide esters via amide bond formation betweena Drug unit and a Linker. This procedure is particularly useful for Drugunits that do not contain free carboxylic group, or for Drugs that havecarboxylic group protected as acid labile esters, preferably adimethoxybenzyl ester.

Drug (Ib) (1.0 eq.) and a suitable cyclic anhydride, preferably glutaricanhydride (1.0 eq.), are diluted with a suitable organic solvent, suchas dichloromethane, and the resulting solution is treated with anorganic base, preferably DIEA (3 eq.). The reaction mixture is allowedto stir under an inert atmosphere, preferably argon, for 24 h, duringwhich time the reaction mixture is monitored using HPLC. The reactionproduct AA1 is isolated using flash chromatography on silica gel. Vacuumdried material AA1 and N,N′-disuccinimidyl carbonate (3 eq.) are dilutedwith a suitable organic solvent, such as dichloromethane, and theresulting solution is treated with an organic base, preferably DIEA (3eq.). The reaction mixture is allowed to stir under an inert atmosphere,preferably argon, for 24 h, during which time the reaction mixture ismonitored using HPLC. The reaction product AA2 is isolated using flashchromatography on silica gel. If necessary, the acid protecting group ofthe Drug-Linker AA2 can now be removed by the appropriate treatment,preferably with 1% TFA in dichloromethane for dimethoxybenzyl ester.

An alternate method of preparing Drug-Linker Compounds is outlined inScheme 14. Using the method of Scheme 14, the Drug is attached to apartial Linker unit (ZA, for example), which does not have a Stretcherunit attached. This provides intermediate AP, which has an Amino Acidunit having an Fmoc-protected N-terminus. The Fmoc group is then removedand the resulting amine intermediate AQ is then attached to a Stretcherunit via a coupling reaction catalyzed using PyBrop or DEPC. Theconstruction of Drug-Linker Compounds containing either a bromoacetamideStretcher AR or a PEG maleimide Stretcher AS is illustrated in Scheme14.

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;and m is an integer ranging from 0-4.

Application of this general strategy for preparation of lysines reactiveN-hydroxysuccinimide ester Linker-Drug is depicted in Scheme 14a.

General Procedure U

Alternative Method of N-Hydroxysuccinimide Ester Linker-Drug Preparation

The intermediate AQ (1 eq.) is suspended in pyridine, and this mixtureis added dropwise to the suspension of disuccinimidyl suberate (5 eq) inpyridine. The reaction mixture is then allowed to stir under an inertatmosphere, preferably argon, for about 4 h, during which time thereaction progress is monitored using HPLC. Pyridine is then removed invacuo, the residue is suspended in a suitable organic solvent, such asdichloromethane, and the Drug-Linker AQ1 is isolated using flashchromatography on silica gel. If necessary, the carboxyl protectinggroup of the Drug can now be removed by the appropriate treatment,preferably with 1% TFA in dichloromethane for dimethoxybenzyl ester.

Methodology useful for the preparation of a Linker unit containing abranched spacer is shown in Scheme 15.

Scheme 15 illustrates the synthesis of a val-cit dipeptide linker havinga maleimide Stretcher unit and a bis(4-hydroxymethyl)styrene (BHMS)unit. The synthesis of the BHMS intermediate (AW) has been improved fromprevious procedures (see, e.g., International Publication No. WO98/13059 to Firestone et al., and Crozet et al., 1985, Tetrahedron Lett.26:5133-5134) and utilizes as starting materials, commercially availablediethyl (4-nitrobenzyl)phosphonate (AT) and commercially available2,2-dimethyl-1,3-dioxan-5-one (AU). Linkers AY and BA can be preparedfrom intermediate AW using the methodology described in Scheme 9.

Dentritic Linkers

The linker may be a dendritic type linker for covalent attachment ofmore than one drug moiety through a branching, multifunctional linkermoiety to a Ligand, such as but not limited to an antibody (see, e.g.,Sun et al., 2002, Bioorganic & Medicinal Chemistry Letters 12:2213-2215;Sun et al., 2003, Bioorganic & Medicinal Chemistry 11:1761-1768).Dendritic linkers can increase the molar ratio of drug to antibody, i.e.loading, which is related to the potency of the Drug-Linker-LigandConjugate. Thus, where a cysteine engineered antibody bears only onereactive cytsteine thiol group, a multitude of drug moieties may beattached through a dendritic linker.

The following exemplary embodiments of dendritic linker reagents allowup to nine nucleophilic drug moiety reagents to be conjugated byreaction with the chloroethyl nitrogen mustard functional groups:

Conjugation of Drug Moieties to Antibodies

Scheme 16 illustrates methodology useful for making Drug-Linker-Ligandconjugates having about 2 to about 4 drugs per antibody. An antibody istreated with a reducing agent, such as dithiothreitol (DTT) to reducesome or all of the cysteine disulfide residues to form highlynucleophilic cysteine thiol groups (—CH₂SH). The partially reducedantibody thus reacts with drug-linker compounds, or linker reagents,with electrophilic functional groups such as maleimide or α-halocarbonyl, according to the conjugation method at page 766 of Klussman etal., 2004, Bioconjugate Chemistry 15(4):765-773.

For example, an antibody, e.g., AC10, dissolved in 500 mM sodium borateand 500 mM sodium chloride at pH 8.0 is treated with an excess of 100 mMdithiothreitol (DTT). After incubation at 37° C. for about 30 minutes,the buffer is exchanged by elution over Sephadex G25 resin and elutedwith PBS with 1 mM DTPA. The thiol/Ab value is checked by determiningthe reduced antibody concentration from the absorbance at 280 nm of thesolution and the thiol concentration by reaction with DTNB (Aldrich,Milwaukee, Wis.) and determination of the absorbance at 412 nm. Thereduced antibody is dissolved in PBS and is chilled on ice. The druglinker, e.g., MC-val-cit-PAB-MMAZ in DMSO, dissolved in acetonitrile andwater at known concentration, is added to the chilled reduced antibodyin PBS. After about one hour, an excess of maleimide is added to quenchthe reaction and cap any unreacted antibody thiol groups. The reactionmixture is concentrated by centrifugal ultrafiltration and the ADC,e.g., AC10-MC-vc-PAB-MMAZ, is purified and desalted by elution throughG25 resin in PBS, filtered through 0.2 μm filters under sterileconditions, and frozen for storage.

A variety of antibody drug conjugates (ADC) can be prepared, with avariety of linkers, and the drug moieties, MMAZ following the protocolsof the Examples, and characterized by HPLC and drug loading assay.

Compositions and Methods of Administration

In other embodiments, described is a composition including an effectiveamount of an Exemplary Compound and/or Exemplary Conjugate and apharmaceutically acceptable carrier or vehicle. For convenience, theDrug units and Drug-Linker Compounds can be referred to as ExemplaryCompounds, while Drug-Ligand Conjugates and Drug-Linker-LigandConjugates can be referred to as Exemplary Conjugates. The compositionsare suitable for veterinary or human administration.

The present compositions can be in any form that allows for thecomposition to be administered to a patient. For example, thecomposition can be in the form of a solid, liquid or gas (aerosol).Typical routes of administration include, without limitation, oral,topical, parenteral, sublingual, rectal, vaginal, ocular, intra-tumor,and intranasal. Parenteral administration includes subcutaneousinjections, intravenous, intramuscular, intrasternal injection orinfusion techniques. In one aspect, the compositions are administeredparenterally. In yet another aspect, the Exemplary Compounds and/or theExemplary Conjugates or compositions are administered intravenously.

Pharmaceutical compositions can be formulated so as to allow anExemplary Compound and/or Exemplary Conjugate to be bioavailable uponadministration of the composition to a patient. Compositions can takethe form of one or more dosage units, where for example, a tablet can bea single dosage unit, and a container of an Exemplary Compound and/orExemplary Conjugate in aerosol form can hold a plurality of dosageunits.

Materials used in preparing the pharmaceutical compositions can benon-toxic in the amounts used. It will be evident to those of ordinaryskill in the art that the optimal dosage of the active ingredient(s) inthe pharmaceutical composition will depend on a variety of factors.Relevant factors include, without limitation, the type of animal (e.g.,human), the particular form of the Exemplary Compound or ExemplaryConjugate, the manner of administration, and the composition employed.

The pharmaceutically acceptable carrier or vehicle can be particulate,so that the compositions are, for example, in tablet or powder form. Thecarrier(s) can be liquid, with the compositions being, for example, anoral syrup or injectable liquid. In addition, the carrier(s) can begaseous or particulate, so as to provide an aerosol composition usefulin, e.g., inhalatory administration.

When intended for oral administration, the composition is preferably insolid or liquid form, where semi-solid, semi-liquid, suspension and gelforms are included within the forms considered herein as either solid orliquid.

As a solid composition for oral administration, the composition can beformulated into a powder, granule, compressed tablet, pill, capsule,chewing gum, wafer or the like form. Such a solid composition typicallycontains one or more inert diluents. In addition, one or more of thefollowing can be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, or gelatin; excipients such asstarch, lactose or dextrins, disintegrating agents such as alginic acid,sodium alginate, Primogel, corn starch and the like; lubricants such asmagnesium stearate or Sterotex; glidants such as colloidal silicondioxide; sweetening agents such as sucrose or saccharin, a flavoringagent such as peppermint, methyl salicylate or orange flavoring, and acoloring agent.

When the composition is in the form of a capsule, e.g., a gelatincapsule, it can contain, in addition to materials of the above type, aliquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

The composition can be in the form of a liquid, e.g., an elixir, syrup,solution, emulsion or suspension. The liquid can be useful for oraladministration or for delivery by injection. When intended for oraladministration, a composition can comprise one or more of a sweeteningagent, preservatives, dye/colorant and flavor enhancer. In a compositionfor administration by injection, one or more of a surfactant,preservative, wetting agent, dispersing agent, suspending agent, buffer,stabilizer and isotonic agent can also be included.

The liquid compositions, whether they are solutions, suspensions orother like form, can also include one or more of the following: sterilediluents such as water for injection, saline solution, preferablyphysiological saline, Ringer's solution, isotonic sodium chloride, fixedoils such as synthetic mono or diglycerides which can serve as thesolvent or suspending medium, polyethylene glycols, glycerin,cyclodextrin, propylene glycol or other solvents; antibacterial agentssuch as benzyl alcohol or methyl paraben; antioxidants such as ascorbicacid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates and agents for the adjustment of tonicity such as sodiumchloride or dextrose. A parenteral composition can be enclosed inampoule, a disposable syringe or a multiple-dose vial made of glass,plastic or other material. Physiological saline is an exemplaryadjuvant. An injectable composition is preferably sterile.

The amount of the Exemplary Compound and/or Exemplary Conjugate that iseffective in the treatment of a particular disorder or condition willdepend on the nature of the disorder or condition, and can be determinedby standard clinical techniques. In addition, in vitro or in vivo assayscan optionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the compositions will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances.

The compositions comprise an effective amount of an Exemplary Compoundand/or Exemplary Conjugate such that a suitable dosage will be obtained.Typically, this amount is at least about 0.01% of an Exemplary Compoundand/or Exemplary Conjugate by weight of the composition. When intendedfor oral administration, this amount can be varied to range from about0.1% to about 80% by weight of the composition. In one aspect, oralcompositions can comprise from about 4% to about 50% of the ExemplaryCompound and/or Exemplary Conjugate by weight of the composition. In yetanother aspect, present compositions are prepared so that a parenteraldosage unit contains from about 0.01% to about 2% by weight of theExemplary Compound and/or Exemplary Conjugate.

For intravenous administration, the composition can comprise from about0.01 to about 100 mg of an Exemplary Compound and/or Exemplary Conjugateper kg of the animal's body weight. In one aspect, the composition caninclude from about 1 to about 100 mg of an Exemplary Compound and/orExemplary Conjugate per kg of the animal's body weight. In anotheraspect, the amount administered will be in the range from about 0.1 toabout 25 mg/kg of body weight of the Exemplary Compound and/or ExemplaryConjugate.

Generally, the dosage of an Exemplary Compound and/or ExemplaryConjugate administered to a patient is typically about 0.01 mg/kg toabout 2000 mg/kg of the animal's body weight. In one aspect, the dosageadministered to a patient is between about 0.01 mg/kg to about 10 mg/kgof the animal's body weight, in another aspect, the dosage administeredto a patient is between about 0.1 mg/kg and about 250 mg/kg of theanimal's body weight, in yet another aspect, the dosage administered toa patient is between about 0.1 mg/kg and about 20 mg/kg of the animal'sbody weight, in yet another aspect the dosage administered is betweenabout 0.1 mg/kg to about 10 mg/kg of the animal's body weight, and inyet another aspect, the dosage administered is between about 1 mg/kg toabout 10 mg/kg of the animal's body weight.

The Exemplary Compounds and/or Exemplary Conjugate or compositions canbe administered by any convenient route, for example by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.).Administration can be systemic or local. Various delivery systems areknown, e.g., encapsulation in liposomes, microparticles, microcapsules,capsules, etc., and can be used to administer an Exemplary Compoundand/or Exemplary Conjugate or composition. In certain embodiments, morethan one Exemplary Compound and/or Exemplary Conjugate or composition isadministered to a patient.

In specific embodiments, it can be desirable to administer one or moreExemplary Compounds and/or Exemplary Conjugate or compositions locallyto the area in need of treatment. This can be achieved, for example, andnot by way of limitation, by local infusion during surgery; topicalapplication, e.g., in conjunction with a wound dressing after surgery;by injection; by means of a catheter; by means of a suppository; or bymeans of an implant, the implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. In one embodiment, administration can be by direct injectionat the site (or former site) of a cancer, tumor or neoplastic orpre-neoplastic tissue. In another embodiment, administration can be bydirect injection at the site (or former site) of a manifestation of anautoimmune disease.

In certain embodiments, it can be desirable to introduce one or moreExemplary Compounds and/or Exemplary Conjugate or compositions into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection. Intraventricular injection can be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir.

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent, or viaperfusion in a fluorocarbon or synthetic pulmonary surfactant.

In yet another embodiment, the Exemplary Compounds and/or ExemplaryConjugate or compositions can be delivered in a controlled releasesystem, such as but not limited to, a pump or various polymericmaterials can be used. In yet another embodiment, a controlled-releasesystem can be placed in proximity of the target of the ExemplaryCompounds and/or Exemplary Conjugate or compositions, e.g., the brain,thus requiring only a fraction of the systemic dose (see, e.g., Goodson,in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138 (1984)). Other controlled-release systems discussed in thereview by Langer (Science 249:1527-1533 (1990)) can be used.

The term “carrier” refers to a diluent, adjuvant or excipient, withwhich an Exemplary Compound and/or Exemplary Conjugate is administered.Such pharmaceutical carriers can be liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.The carriers can be saline, gum acacia, gelatin, starch paste, talc,keratin, colloidal silica, urea, and the like. In addition, auxiliary,stabilizing, thickening, lubricating and coloring agents can be used. Inone embodiment, when administered to a patient, the Exemplary Compoundand/or Exemplary Conjugate or compositions and pharmaceuticallyacceptable carriers are sterile. Water is an exemplary carrier when theExemplary Compounds and/or Exemplary Conjugates are administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical carriers also includeexcipients such as starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The present compositions, if desired, canalso contain minor amounts of wetting or emulsifying agents, or pHbuffering agents.

The present compositions can take the form of solutions, suspensions,emulsion, tablets, pills, pellets, capsules, capsules containingliquids, powders, sustained-release formulations, suppositories,emulsions, aerosols, sprays, suspensions, or any other form suitable foruse. Other examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin.

In an embodiment, the Exemplary Compounds and/or Exemplary Conjugatesare formulated in accordance with routine procedures as a pharmaceuticalcomposition adapted for intravenous administration to animals,particularly human beings. Typically, the carriers or vehicles forintravenous administration are sterile isotonic aqueous buffersolutions. Where necessary, the compositions can also include asolubilizing agent. Compositions for intravenous administration canoptionally comprise a local anesthetic such as lignocaine to ease painat the site of the injection. Generally, the ingredients are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent. Where an Exemplary Compound and/or Exemplary Conjugateis to be administered by infusion, it can be dispensed, for example,with an infusion bottle containing sterile pharmaceutical grade water orsaline. Where the Exemplary Compound and/or Exemplary Conjugate isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients can be mixed prior toadministration.

Compositions for oral delivery can be in the form of tablets, lozenges,aqueous or oily suspensions, granules, powders, emulsions, capsules,syrups, or elixirs, for example. Orally administered compositions cancontain one or more optionally agents, for example, sweetening agentssuch as fructose, aspartame or saccharin; flavoring agents such aspeppermint, oil of wintergreen, or cherry; coloring agents; andpreserving agents, to provide a pharmaceutically palatable preparation.Moreover, where in tablet or pill form, the compositions can be coatedto delay disintegration and absorption in the gastrointestinal tractthereby providing a sustained action over an extended period of time.Selectively permeable membranes surrounding an osmotically activedriving compound are also suitable for orally administered compounds. Inthese later platforms, fluid from the environment surrounding thecapsule is imbibed by the driving compound, which swells to displace theagent or agent composition through an aperture. These delivery platformscan provide an essentially zero order delivery profile as opposed to thespiked profiles of immediate release formulations. A time-delay materialsuch as glycerol monostearate or glycerol stearate can also be used.

The compositions can be intended for topical administration, in whichcase the carrier may be in the form of a solution, emulsion, ointment orgel base. If intended for transdermal administration, the compositioncan be in the form of a transdermal patch or an iontophoresis device.Topical formulations can comprise a concentration of an ExemplaryCompound and/or Exemplary Conjugate of from about 0.05% to about 50% w/v(weight per unit volume of composition), in another aspect, from 0.1% to10% w/v.

The composition can be intended for rectal administration, in the form,e.g., of a suppository which will melt in the rectum and release theExemplary Compound and/or Exemplary Conjugate.

The composition can include various materials that modify the physicalform of a solid or liquid dosage unit. For example, the composition caninclude materials that form a coating shell around the activeingredients. The materials that form the coating shell are typicallyinert, and can be selected from, for example, sugar, shellac, and otherenteric coating agents. Alternatively, the active ingredients can beencased in a gelatin capsule.

The compositions can consist of gaseous dosage units, e.g., it can be inthe form of an aerosol. The term aerosol is used to denote a variety ofsystems ranging from those of colloidal nature to systems consisting ofpressurized packages. Delivery can be by a liquefied or compressed gasor by a suitable pump system that dispenses the active ingredients.

Whether in solid, liquid or gaseous form, the present compositions caninclude a pharmacological agent used in the treatment of cancer, anautoimmune disease or an infectious disease.

Therapeutics Uses of the Exemplary Conjugates

The Exemplary Compounds and/or Exemplary Conjugates are useful fortreating cancer, an autoimmune disease or an infectious disease in apatient.

Treatment of Cancer

The Exemplary Compounds and/or Exemplary Conjugates are useful forinhibiting the multiplication of a tumor cell or cancer cell, causingapoptosis in a tumor or cancer cell, or for treating cancer in apatient. The Exemplary Compounds and/or Exemplary Conjugates can be usedaccordingly in a variety of settings for the treatment of animalcancers. The Drug-Linker-Ligand Conjugates can be used to deliver a Drugor Drug unit to a tumor cell or cancer cell. Without being bound bytheory, in one embodiment, the Ligand unit of an Exemplary Conjugatebinds to or associates with a cancer-cell or a tumor-cell-associatedantigen, and the Exemplary Conjugate can be taken up (internalized)inside a tumor cell or cancer cell through receptor-mediatedendocytosis. The antigen can be attached to a tumor cell or cancer cellor can be an extracellular matrix protein associated with the tumor cellor cancer cell. Once inside the cell, one or more specific peptidesequences within the Linker unit are hydrolytically cleaved by one ormore tumor-cell or cancer-cell-associated proteases, resulting inrelease of a Drug or a Drug-Linker Compound. The released Drug orDrug-Linker Compound is then free to migrate within the cell and inducecytotoxic or cytostatic activities. The Drug-Linker-Ligand conjugatealso can be cleaved by intracellular protease to release the Drugmoiety, the Drug-Linker compound, and/or an active fragment of theDrug-Linker-Ligand conjugate (e.g., cystyl-Linker-Drug). In analternative embodiment, the Drug or Drug unit is cleaved from theExemplary Conjugate outside the tumor cell or cancer cell, and the Drugor Drug-Linker Compound subsequently penetrates the cell. In analternative embodiment, the Drug or Drug unit is cleaved from theExemplary Conjugate outside the tumor cell or cancer cell, and the Drugor Drug-Linker Compound subsequently penetrates the cell.

In one embodiment, the Ligand unit binds to the tumor cell or cancercell.

In another embodiment, the Ligand unit binds to a tumor cell or cancercell antigen which is on the surface of the tumor cell or cancer cell.

In another embodiment, the Ligand unit binds to a tumor cell or cancercell antigen which is an extracellular matrix protein associated withthe tumor cell or cancer cell.

The specificity of the Ligand unit for a particular tumor cell or cancercell can be important for determining those tumors or cancers that aremost effectively treated. For example, Exemplary Conjugates having aBR96 Ligand unit can be useful for treating antigen positive carcinomasincluding those of the lung, breast, colon, ovaries, and pancreas.Exemplary Conjugates having an anti-CD30 or an anti-CD40 Ligand unit canbe useful for treating hematologic malignancies.

Other particular types of cancers that can be treated with ExemplaryConjugates include, but are not limited to, those disclosed in Table 1:

TABLE 1 Solid tumors, including but not limited to: fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer,kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovariancancer, prostate cancer, esophogeal cancer, stomach cancer, oral cancer,nasal cancer, throat cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterinecancer, testicular cancer, small cell lung carcinoma, bladder carcinoma,lung cancer, epithelial carcinoma, glioma, glioblastoma multiforme,astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skincancer, melanoma, neuroblastoma, retinoblastoma blood-borne cancers,including but not limited to: acute lymphoblastic leukemia “ALL”, acutelymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia,acute myeloblastic leukemia “AML”, acute promyelocytic leukemia “APL”,acute monoblastic leukemia, acute erythroleukemic leukemia, acutemegakaryoblastic leukemia, acute myelomonocytic leukemia, acutenonlymphocyctic leukemia, acute undifferentiated leukemia, chronicmyelocytic leukemia “CML”, chronic lymphocytic leukemia “CLL”, hairycell leukemia, multiple myeloma acute and chronic leukemias:lymphoblastic, myelogenous, lymphocytic, myelocytic leukemias Lymphomas:Hodgkin's disease, non-Hodgkin's Lymphoma, Multiple myeloma,Waldenström's macroglobulinemia, Heavy chain disease, Polycythemia vera

The Exemplary Conjugates provide conjugation-specific tumor or cancertargeting, thus reducing general toxicity of these compounds. The Linkerunits stabilize the Exemplary Conjugates in blood, yet are cleavable bytumor-specific proteases within the cell, liberating a Drug.

Multi-Modality Therapy for Cancer

Cancers, including, but not limited to, a tumor, metastasis, or otherdisease or disorder characterized by uncontrolled cell growth, can betreated or prevented by administration of an Exemplary Conjugate and/oran Exemplary Compound.

In other embodiments, methods for treating or preventing cancer areprovided, including administering to a patient in need thereof aneffective amount of an Exemplary Conjugate and a chemotherapeutic agent.In one embodiment the chemotherapeutic agent is that with whichtreatment of the cancer has not been found to be refractory. In anotherembodiment, the chemotherapeutic agent is that with which the treatmentof cancer has been found to be refractory. The Exemplary Conjugates canbe administered to a patient that has also undergone surgery astreatment for the cancer.

In some embodiment, the patient is also receives an additionaltreatment, such as radiation therapy. In a specific embodiment, theExemplary Conjugate is administered concurrently with thechemotherapeutic agent or with radiation therapy. In another specificembodiment, the chemotherapeutic agent or radiation therapy isadministered prior or subsequent to administration of an ExemplaryConjugates, in one aspect at least an hour, five hours, 12 hours, a day,a week, a month, in further aspects several months (e.g., up to threemonths), prior or subsequent to administration of an ExemplaryConjugate.

A chemotherapeutic agent can be administered over a series of sessions.Any one or a combination of the chemotherapeutic agents listed in Table4 can be administered. With respect to radiation, any radiation therapyprotocol can be used depending upon the type of cancer to be treated.For example, but not by way of limitation, x-ray radiation can beadministered; in particular, high-energy megavoltage (radiation ofgreater that 1 MeV energy) can be used for deep tumors, and electronbeam and orthovoltage x-ray radiation can be used for skin cancers.Gamma-ray emitting radioisotopes, such as radioactive isotopes ofradium, cobalt and other elements, can also be administered.

Additionally, methods of treatment of cancer with an Exemplary Compoundand/or Exemplary Conjugate are provided as an alternative tochemotherapy or radiation therapy where the chemotherapy or theradiation therapy has proven or can prove too toxic, e.g., results inunacceptable or unbearable side effects, for the subject being treated.The animal being treated can, optionally, be treated with another cancertreatment such as surgery, radiation therapy or chemotherapy, dependingon which treatment is found to be acceptable or bearable.

The Exemplary Compounds and/or Exemplary Conjugates can also be used inan in vitro or ex vivo fashion, such as for the treatment of certaincancers, including, but not limited to leukemias and lymphomas, suchtreatment involving autologous stem cell transplants. This can involve amulti-step process in which the animal's autologous hematopoietic stemcells are harvested and purged of all cancer cells, the animal'sremaining bone-marrow cell population is then eradicated via theadministration of a high dose of an Exemplary Compound and/or ExemplaryConjugate with or without accompanying high dose radiation therapy, andthe stem cell graft is infused back into the animal. Supportive care isthen provided while bone marrow function is restored and the animalrecovers.

Multi-Drug Therapy for Cancer

Methods for treating cancer include administering to a patient in needthereof an effective amount of an Exemplary Conjugate and anothertherapeutic agent that is an anti-cancer agent. An “anti-cancer agent”or a “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer.

Suitable anticancer agents include, but are not limited to,methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine,hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas,cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, topotecan,nitrogen mustards, cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan,camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin,dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine,vincristine, vinorelbine, paclitaxel, and docetaxel.

Examples of chemotherapeutic agents include alkylating agents such asthiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such asbusulfan, improsulfan, piposulfan and treosulfan; aziridines such asbenzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; TLK 286 (TELCYTA™); acetogenins (especiallybullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol,MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; acamptothecin (including the synthetic analogue topotecan (HYCAMTIN®),CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin,crisnatol; and 9-aminocamptothecin); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); podophyllotoxin; podophyllinic acid; teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, and uracil mustard;nitrosureas such as carmustine (BCNU), chlorozotocin, fotemustine,lomustine, nimustine, and ranimustine; triazines, such as dacarbazine;bisphosphonates, such as clodronate; antibiotics such as the enediyneantibiotics (e.g., calicheamicin, especially calicheamicin gamma1I andcalicheamicin omegaI1 (see, e.g., Agnew, 1994, Chem Intl. Ed. Engl.33:183-186) and anthracyclines such as annamycin, AD 32, alcarubicin,daunorubicin, dexrazoxane, DX-52-1, epirubicin, GPX-100, idarubicin,pirarubicin, zorubicin, mitoxantrone, KRN5500, menogaril, dynemicin,including dynemicin A, an esperamicin, neocarzinostatin chromophore andrelated chromoprotein enediyne antibiotic chromophores, aclacinomysins,actinomycin, authramycin, azaserine, bleomycins (e.g., A2 or B2),cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis,dactinomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomaldoxorubicin, and deoxydoxorubicin), EICAR, esorubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, ribavirin,rodorubicin, streptonigrin, streptozocin, tiazofurin, tubercidin,ubenimex, zinostatin, and zorubicin; folic acid analogues such asdenopterin, pteropterin, and trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, cytoarabinoside, dideoxyuridine, doxifluridine, enocitabine,floxuridine and fludarabine; androgens such as calusterone,dromostanolone propionate, epitiostanol, mepitiostane, and testolactone;anti-adrenals such as aminoglutethimide, mitotane, and trilostane; folicacid replenisher such as folinic acid (leucovorin); aceglatone;anti-folate anti-neoplastic agents such as ALIMTA®, LY231514 pemetrexed,dihydrofolate reductase inhibitors such as methotrexate andtrimetrexate, anti-metabolites such as 5-fluorouracil (5-FU) and itsprodrugs such as UFT, S-1 and capecitabine, and thymidylate synthaseinhibitors and glycinamide ribonucleotide formyltransferase inhibitorssuch as raltitrexed (TOMUDEX™, TDX); inhibitors of dihydropyrimidinedehydrogenase such as eniluracil; aldophosphamide glycoside;aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate;defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate;an epothilone; etoglucid; gallium nitrate; hydroxyurea; defereoxamine;lentinan; lonidainine; maytansinoids such as maytansine andansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene,Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine(ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol;mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”);cyclophosphamide; thiotepa; taxoids and taxanes, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; platinum; platinumanalogs or platinum-based analogs such as cisplatin, oxaliplatin andcarboplatin; vinblastine (VELBAN®); etoposide (VP-16); ifosfamide;mitoxantrone; vincristine (ONCOVIN®); vinblastine; vindesine;vinorelbine; vinca alkaloid; vinorelbine (NAVELBINE®); novantrone;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomeraseinhibitor RPS 2000; difluoromethylornithine (DMFO); MDR inhibitors suchas verapamil; retinoids such as retinoic acid; cell cycle inhibitors,such as staurosporine; Lovastatin; REVLIMID (lenalidomide); THALAMID(thalidomide); VELADE (bortezomib); pharmaceutically acceptable salts,acids or derivatives of any of the above; as well as combinations of twoor more of the above such as CHOP, an abbreviation for a combinedtherapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone,and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin(ELOXATIN™) combined with 5-FU and leucovorin.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON® toremifene; aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol,MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide(e.g., leuprolide acetate), and goserelin; as well as troxacitabine (a1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides,particularly those that inhibit expression of genes in signalingpathways implicated in abherant cell proliferation, such as, forexample, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor(EGF-R); vaccines such as gene therapy vaccines, for example,ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN®rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

Also included in this definition are Vitamin D3 analogs, such as EB1089, CB 1093 and KH 1060; and Photodynamic therapies, such asvertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4,demethoxy-hypocrellin A and 2BA-2-DMHA.

Treatment of Autoimmune Diseases

The Exemplary Conjugates are useful for killing or inhibiting thereplication of a cell that produces an autoimmune disease or fortreating an autoimmune disease. The Exemplary Conjugates can be usedaccordingly in a variety of settings for the treatment of an autoimmunedisease in a patient. The Drug-Linker-Ligand Conjugates can be used todeliver a Drug to a target cell. Without being bound by theory, in oneembodiment, the Drug-Linker-Ligand Conjugate associates with an antigenon the surface of a target cell, and the Exemplary Conjugate is thentaken up inside a target-cell through receptor-mediated endocytosis.Once inside the cell, one or more specific peptide sequences within theLinker unit are enzymatically or hydrolytically cleaved, resulting inrelease of a Drug. The released Drug is then free to migrate in thecytosol and induce cytotoxic or cytostatic activities. TheDrug-Linker-Ligand conjugate also can be cleaved by intracellularprotease to release the Drug moiety, the Drug-Linker compound, and/or anactive fragment of the Drug-Linker-Ligand conjugate (e.g.,cystyl-Linker-Drug). In an alternative embodiment, the Drug is cleavedfrom the Exemplary Conjugate outside the target cell, and the Drugsubsequently penetrates the cell.

In one embodiment, the Ligand unit binds to an autoimmune antigen. Inone aspect, the antigen is on the surface of a cell involved in anautoimmune condition.

In another embodiment, the Ligand unit binds to an autoimmune antigenwhich is on the surface of a cell.

In one embodiment, the Ligand binds to activated lymphocytes that areassociated with the autoimmune disease state.

In a further embodiment, the Exemplary Conjugates kill or inhibit themultiplication of cells that produce an autoimmune antibody associatedwith a particular autoimmune disease.

Particular types of autoimmune diseases that can be treated with theExemplary Conjugates include, but are not limited to, Th2 lymphocyterelated disorders (e.g., atopic dermatitis, atopic asthma,rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemicsclerosis, and graft versus host disease); Th1 lymphocyte-relateddisorders (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis,Sjogren's syndrome, Hashimoto's thyroiditis, Grave's disease, primarybiliary cirrhosis, Wegener's granulomatosis, and tuberculosis);activated B lymphocyte-related disorders (e.g., systemic lupuserythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type Idiabetes); and those disclosed in Table 3.

TABLE 3 Active Chronic Hepatitis, Addison's Disease, AllergicAlveolitis, Allergic Reaction, Allergic Rhinitis, Alport's Syndrome,Anaphlaxis, Ankylosing Spondylitis, Anti-phosholipid Syndrome,Arthritis, Ascariasis, Aspergillosis, Atopic Allergy, AtropicDermatitis, Atropic Rhinitis, Behcet's Disease, Bird-Fancier's Lung,Bronchial Asthma, Caplan's Syndrome, Cardiomyopathy, Celiac Disease,Chagas' Disease, Chronic Glomerulonephritis, Cogan's Syndrome, ColdAgglutinin Disease, Congenital Rubella Infection, CREST Syndrome,Crohn's Disease, Cryoglobulinemia, Cushing's Syndrome, Dermatomyositis,Discoid Lupus, Dressler's Syndrome, Eaton-Lambert Syndrome, EchovirusInfection, Encephalomyelitis, Endocrine opthalmopathy, Epstein-BarrVirus Infection, Equine Heaves, Erythematosis, Evan's Syndrome, Felty'sSyndrome, Fibromyalgia, Fuch's Cyclitis, Gastric Atrophy,Gastrointestinal Allergy, Giant Cell Arteritis, Glomerulonephritis,Goodpasture's Syndrome, Graft v. Host Disease, Graves' Disease,Guillain-Barre Disease, Hashimoto's Thyroiditis, Hemolytic Anemia,Henoch-Schonlein Purpura, Idiopathic Adrenal Atrophy, IdiopathicPulmonary Fibritis, IgA Nephropathy, Inflammatory Bowel Diseases,Insulin-dependent Diabetes Mellitus, Juvenile Arthritis, JuvenileDiabetes Mellitus (Type I), Lambert-Eaton Syndrome, Laminitis, LichenPlanus, Lupoid Hepatitis, Lupus, Lymphopenia, Meniere's Disease, MixedConnective Tissue Disease, Multiple Sclerosis, Myasthenia Gravis,Pernicious Anemia, Polyglandular Syndromes, Presenile Dementia, PrimaryAgammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis, PsoriaticArthritis, Raynauds Phenomenon, Recurrent Abortion, Reiter's Syndrome,Rheumatic Fever, Rheumatoid Arthritis, Sampter's Syndrome,Schistosomiasis, Schmidt's Syndrome, Scleroderma, Shulman's Syndrome,Sjorgen's Syndrome, Stiff-Man Syndrome, Sympathetic Ophthalmia, SystemicLupus Erythematosis, Takayasu's Arteritis, Temporal Arteritis,Thyroiditis, Thrombocytopenia, Thyrotoxicosis, Toxic EpidermalNecrolysis, Type B Insulin Resistance, Type I Diabetes Mellitus,Ulcerative Colitis, Uveitis, Vitiligo, Waldenstrom's Macroglobulemia,Wegener's Granulomatosis

Multi-Drug Therapy of Autoimmune Diseases

Methods for treating an autoimmune disease are also disclosed includingadministering to a patient in need thereof an effective amount of anExemplary Conjugate and another therapeutic agent known for thetreatment of an autoimmune disease. In one embodiment, theanti-autoimmune disease agent includes, but is not limited to, agentslisted in Table 4.

TABLE 4 cyclosporine, cyclosporine A, mycophenylate mofetil, sirolimus,tacrolimus, enanercept, prednisone, azathioprine, methotrexate,cyclophosphamide, prednisone, aminocaproic acid, chloroquine,hydroxychloroquine, hydrocortisone, dexamethasone, chlorambucil, DHEA,danazol, bromocriptine, meloxicam, infliximab

Treatment of Infectious Diseases

The Exemplary Conjugates are useful for killing or inhibiting themultiplication of a cell that produces an infectious disease or fortreating an infectious disease. The Exemplary Conjugates can be usedaccordingly in a variety of settings for the treatment of an infectiousdisease in a patient. The Drug-Linker-Ligand Conjugates can be used todeliver a Drug to a target cell. In one embodiment, the Ligand unitbinds to the infectious disease cell.

In one embodiment, the Conjugates kill or inhibit the multiplication ofcells that produce a particular infectious disease.

Particular types of infectious diseases that can be treated with theExemplary Conjugates include, but are not limited to, those disclosed inTable 5.

TABLE 5 Bacterial Diseases: Diphtheria, Pertussis, Occult Bacteremia,Urinary Tract Infection, Gastroenteritis, Cellulitis, Epiglottitis,Tracheitis, Adenoid Hypertrophy, Retropharyngeal Abcess, Impetigo,Ecthyma, Pneumonia, Endocarditis, Septic Arthritis, Pneumococca,Peritonitis, Bactermia, Meningitis, Acute Purulent Meningitis,Urethritis, Cervicitis, Proctitis, Pharyngitis, Salpingitis,Epididymitis, Gonorrhea, Syphilis, Listeriosis, Anthrax, Nocardiosis,Salmonella, Typhoid Fever, Dysentery, Conjunctivitis, Sinusitis,Brucellosis, Tullaremia, Cholera, Bubonic Plague, Tetanus, NecrotizingEnteritis, Actinomycosis, Mixed Anaerobic Infections, Syphilis,Relapsing Fever, Leptospirosis, Lyme Disease, Rat Bite Fever,Tuberculosis, Lymphadenitis, Leprosy, Chlamydia, Chlamydial Pneumonia,Trachoma, Inclusion Conjunctivitis Systemic Fungal Diseases:Histoplamosis, Coccidiodomycosis, Blastomycosis, Sporotrichosis,Cryptococcsis, Systemic Candidiasis, Aspergillosis, Mucormycosis,Mycetoma, Chromomycosis Rickettsial Diseases: Typhus, Rocky MountainSpotted Fever, Ehrlichiosis, Eastern Tick-Borne Rickettsioses,Rickettsialpox, Q Fever, Bartonellosis Parasitic Diseases: Malaria,Babesiosis, African Sleeping Sickness, Chagas' Disease, Leishmaniasis,Dum-Dum Fever, Toxoplasmosis, Meningoencephalitis, Keratitis,Entamebiasis, Giardiasis, Cryptosporidiasis, Isosporiasis,Cyclosporiasis, Microsporidiosis, Ascariasis, Whipworm Infection,Hookworm Infection, Threadworm Infection, Ocular Larva Migrans,Trichinosis, Guinea Worm Disease, Lymphatic Filariasis, Loiasis, RiverBlindness, Canine Heartworm Infection, Schistosomiasis, Swimmer's Itch,Oriental Lung Fluke, Oriental Liver Fluke, Fascioliasis,Fasciolopsiasis, Opisthorchiasis, Tapeworm Infections, Hydatid Disease,Alveolar Hydatid Disease Viral Diseases: Measles, Subacute sclerosingpanencephalitis, Common Cold, Mumps, Rubella, Roseola, Fifth Disease,Chickenpox, Respiratory syncytial virus infection, Croup, Bronchiolitis,Infectious Mononucleosis, Poliomyelitis, Herpangina, Hand-Foot-and-MouthDisease, Bornholm Disease, Genital Herpes, Genital Warts, AsepticMeningitis, Myocarditis, Pericarditis, Gastroenteritis, AcquiredImmunodeficiency Syndrome (AIDS), Human Immunodeficiency Virus (HIV),Reye's Syndrome, Kawasaki Syndrome, Influenza, Bronchitis, Viral“Walking” Pneumonia, Acute Febrile Respiratory Disease, Acutepharyngoconjunctival fever, Epidemic keratoconjunctivitis, HerpesSimplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2), Shingles,Cytomegalic Inclusion Disease, Rabies, Progressive MultifocalLeukoencephalopathy, Kuru, Fatal Familial Insomnia, Creutzfeldt-JakobDisease, Gerstmann-Straussler-Scheinker Disease, Tropical SpasticParaparesis, Western Equine Encephalitis, California Encephalitis, St.Louis Encephalitis, Yellow Fever, Dengue, Lymphocytic choriomeningitis,Lassa Fever, Hemorrhagic Fever, Hantvirus Pulmonary Syndrome, MarburgVirus Infections, Ebola Virus Infections, Smallpox

Multi-Drug Therapy of Infectious Diseases

Methods for treating an infectious disease are disclosed includingadministering to a patient in need thereof an Exemplary Conjugate andanother therapeutic agent that is an anti-infectious disease agent. Inone embodiment, the anti-infectious disease agent is, but not limitedto, agents listed in Table 6.

TABLE 6 β-Lactam Antibiotics: Penicillin G, Penicillin V, Cloxacilliin,Dicloxacillin, Methicillin, Nafcillin, Oxacillin, Ampicillin,Amoxicillin, Bacampicillin, Azlocillin, Carbenicillin, Mezlocillin,Piperacillin, Ticarcillin Aminoglycosides: Amikacin, Gentamicin,Kanamycin, Neomycin, Netilmicin, Streptomycin, Tobramycin Macrolides:Azithromycin, Clarithromycin Erythromycin, Lincomycin, ClindamycinTetracyclines: Demeclocycline, Doxycycline, Minocycline,Oxytetracycline, Tetracycline Quinolones: Cinoxacin, Nalidixic AcidFluoroquinolones: Ciprofloxacin, Enoxacin, Grepafloxacin, Levofloxacin,Lomefloxacin, Norfloxacin, Ofloxacin, Sparfloxacin, TrovafloxicinPolypeptides: Bacitracin, Colistin, Polymyxin B Sulfonamides:Sulfisoxazole, Sulfamethoxazole, Sulfadiazine, Sulfamethizole,Sulfacetamide Miscellaneous Antibacterial Agents: Trimethoprim,Sulfamethazole, Chloramphenicol, Vancomycin, Metronidazole,Quinupristin, Dalfopristin, Rifampin, Spectinomycin, NitrofurantoinAntiviral Agents: General Antiviral Agents: Idoxuradine, Vidarabine,Trifluridine, Acyclovir, Famcicyclovir, Pencicyclovir, Valacyclovir,Gancicyclovir, Foscarnet, Ribavirin, Amantadine, Rimantadine, Cidofovir,Antisense Oligonucleotides, Immunoglobulins, Inteferons Drugs for HIVinfection: Tenofovir, Emtricitabine, Zidovudine, Didanosine,Zalcitabine, Stavudine, Lamivudine, Nevirapine, Delavirdine, Saquinavir,Ritonavir, Indinavir, Nelfinavir

EXAMPLES Example 1 Preparation of Compound AB

Fmoc-val-cit-PAB-OH (14.61 g, 24.3 mmol, 1.0 eq.; see, e.g., U.S. Pat.No. 6,214,345 to Firestone et al.) was diluted with DMF (120 mL, 0.2 M)and to this solution was added a diethylamine (60 mL). The reaction wasmonitored by HPLC and found to be complete in 2 h. The reaction mixturewas concentrated and the resulting residue was precipitated using ethylacetate (ca. 100 mL) under sonication over for 10 min. Ether (200 mL)was added and the precipitate was further sonicated for 5 min. Thesolution was allowed to stand for 30 min. without stirring and was thenfiltered and dried under high vacuum to provide Val-cit-PAB-OH, whichwas used in the next step without further purification. Yield: 8.84 g(96%). Val-cit-PAB-OH (8.0 g, 21 mmol) was diluted with DMF (110 mL) andthe resulting solution was treated with MC-OSu (Willner et al., 1993,Bioconjugate Chem. 4:521; 6.5 g, 21 mmol, 1.0 eq.). The reaction wascomplete according to HPLC after 2 h. The reaction mixture wasconcentrated and the resulting oil was precipitated using ethyl acetate(50 mL). After sonicating for 15 min, ether (400 mL) was added and themixture was sonicated further until all large particles were broken up.The solution was then filtered and the solid dried to provide anoff-white solid intermediate. Yield: 11.63 g (96%); ES-MS m/z 757.9[M−H]

The off-white solid intermediate (8.0 g, 14.0 mmol) was diluted with DMF(120 mL, 0.12 M) and to the resulting solution was addedbis(4-nitrophenyl)carbonate (8.5 g, 28.0 mmol, 2.0 eq.) and DIEA (3.66mL, 21.0 mmol, 1.5 eq.). The reaction was complete in 1 h according toHPLC. The reaction mixture was concentrated to provide an oil that isprecipitated with EtOAc, and then triturated with EtOAc (ca. 25 mL). Thesolute was further precipitated with ether (ca. 200 mL) and trituratedfor 15 min. The solid was filtered and dried under high vacuum toprovide Compound AB which is 93% pure according to HPLC and used in thenext step without further purification. Yield: 9.7 g (94%).

Example 2 Preparation of Compounds MMAZ by Solid Phase Synthesis

Fmoc-Aminoacid-2-Chlorotrityl Resins (SP1) were prepared according togeneral Procedure SP(a). The following examples illustrate thepreparation of certain resins.

Fmoc-2-chloro-Phe-2-Chlorotrityl Resin (SP1-z)

Fmoc-2-chloro-L-phenylalanine (354 mg, 0.84 mmol) was dissolved inanhydrous CH₂Cl₂ (4-mL) and DIEA (585 μL, 3.36 mmol, 4 equiv). Theresulting solution was added to a 10-mL syringe containing2-Chlorotrityl chloride resin (500 mg, 0.70 mmol, 1.4 mmol/g). Themixture was agitated for 6 hours at room temperature. The resin wasfiltered, washed with DCM/MeOH/DIEA (17:2:1; 4×5 mL), MeOH (1×5 mL), DCM(4×5 mL), DMF (4×5 mL), DCM (2×5 mL) and ethyl ether (4×5 mL), and wasdried in-vacuo for 2 h. The resin was then left under vacuum overnight.Loading was determined by Fmoc-quantitation. A known quantity (4.4 mg)2-Chloro-Phe-2-Chlorotrityl resin was weighed into a 10-mL volumetricflask. To the flask was transferred 20% piperidine/DMF (2-mL). Themixture was allowed to cleave for 1 h, with occasional agitation byhand. To the flask was transferred DMF (8-mL) to bring the total volumeto 10-mL. A blank solution was prepared with 10-mL of 20% piperidine/DMFin a 10-mL volumetric flask. The spectrophotometer was zeroed with theblank solution. The absorbance was measured at 301 nm and the loadinglevel was given by:Loading (mmol/g)=A ₃₀₁×10 mL/7800×wtA₃₀₁ is the absorbance at 301 nm; 7800 is the extinction coefficient ofthe piperidine-fluorenone adduct, and wt is the weight of resin used inmilligrams. Fmoc quantitation is generally performed in duplicate.Loading level of the Fmoc-2-Chloro-Phe-2-Chlorotrityl resin wasdetermined as 0.612 mmol/g.

Fmoc-Me-Phe-2-Chlorotrityl Resin (SP1-b)

Fmoc-Me-L-phenylalanine (337 mg, 0.84 mmol) was loaded onto2-Chlorotrityl Chloride resin as described in General Procedure SP(a).The loading level of the Fmoc-Me-L-Phe-2-Chlorotrityl resin wasdetermined to be 0.4908 mmol/g.

Fmoc-Tic-2-Chlorotrityl Resin (SP1-c)

Fmoc-Tic-OH (335 mg, 0.84 mmol) was loaded onto 2-Chlorotrityl Chlorideresin as described in General Procedure SP(a). The loading level of theFmoc-Tic-2-Chlorotrityl resin was determined to be 0.638 mmol/g.

Fmoc-L-β-homophe-2-Chlorotrityl Resin (SP1-d)

Fmoc-L-β-homophenylalanine (337 mg, 0.84 mmol) was loaded onto2-Chlorotrityl Chloride resin as described in General Procedure SP(a).The loading level of the Fmoc-L-β-homophe-2-chlorotrityl resin wasdetermined to be 0.579 mmol/g.

Boc-p-Amino-Phe(Fmoc)-2-Chlorotrityl Resin (SP1-e)

Boc-p-Amino-Phe(Fmoc)-OH (704 mg, 0.70 mmol) was loaded onto2-Chlorotrityl Chloride resin as described in General Procedure SP(a).The loading level of the Boc-p-Amino-Phe(Fmoc)-2-chlorotrityl resin wasdetermined as 0.650 mmol/g.

Fmoc-3-cyclohexyl-L-Ala-2-Chlorotrityl Resin (SP1-f)

Fmoc-3-cyclohexyl-L-alanine (550 mg, 0.70 mmol) was loaded onto2-Chlorotrityl Chloride resin as described in General Procedure SP(a).The loading level of the Fmoc-3-cyclohexyl-L-Ala-2-chlorotrityl resinwas determined to be 0.660 mmol/g.

Fmoc-L-4-Thiazolylalanine-2-Chlorotrityl Resin (SP1-g)

Fmoc-L-4-Thiazolylalanine (552 mg, 0.70 mmol) was loaded onto2-Chlorotrityl Chloride resin as described in General Procedure SP(a).The loading level of the Fmoc-L-4-Thiazolylalanine-2-Chlorotrityl resinwas determined to be 0.790 mmol/g.

Fmoc-3-(3-pyridyl)-L-Ala-2-Chlorotrityl Resin (SP1-h)

Fmoc-3-(3-pyridyl)-L-Alanine (543 mg, 0.70 mmol) was loaded onto2-Chlorotrityl Chloride resin as described in General Procedure SP(a).Loading level of the Fmoc-3-(3-pyridyl)-L-Ala-2-Chlorotrityl resin wasdetermined to be 0.790 mmol/g.

Fmoc quantitation of commercially available pre-loaded resins wasperformed according to General Procedure SP(b)

H-Leu-2-Chlorotrityl Resin (SP1-i)

Fmoc-Cl (259 mg, 1 mmol) was dissolved in anhydrous CH₂Cl₂ (2-mL) tomake a 0.5M working solution. The solution was transferred to a 3-mLplastic syringe containing H-Leu-2-Chlorotrityl resin (25 mg, 0.86mmol/g, 0.0215 mmol). The mixture was agitated for 2 hours. The resinwas filtered and washed with DMF (2×5 mL), CH₂Cl₂ (2×5 mL), and ethylether (2×5 mL), and dried in-vacuo for 2 hours. The resin was tested bythe Kaiser amine test. Upon negative results (free amine fullyprotected), Fmoc quantitation was performed to obtain the loading level,as described in General Procedure SP(a). The loading level of theH-Leu-2-Chlorotrityl resin was determined to be 0.85 mmol/g.

H-Met-2-Chlorotrityl Resin (SP1-j)

H-Met-2-Chlorotrityl resin (25 mg, 0.64 mmol/g, 0.016 mmol) was acylatedwith excess Fmoc-Cl (259 mg, 1 mmol), as described in General ProcedureSP(b). The loading level of the H-Met-2-Chlorotrityl resin wasdetermined to be 0.27 mmol/g.

H-Trp(Boc)-2-Chlorotrityl Resin (SP1-k)

H-Trp(Boc)-2-Chlorotrityl Resin (25 mg, 0.74 mmol/g, 0.033 mmol) wasacylated with excess Fmoc-Cl (259 mg, 1 mmol), as described in GeneralProcedure SP(b). The loading level of the H-Trp(Boc)-2-Chlorotritylresin was determined to be 0.70 mmol/g.

H-Glu(OtBu)-2-Chlorotrityl Resin (SP1-l)

H-Glu(OtBu)-2-Chlorotrityl resin (25 mg, 0.90 mmol/g, 0.022 mmol) wasacylated with excess Fmoc-Cl (259 mg, 1 mmol), as described in GeneralProcedure SP(b). The loading level of the H-Glu(OtBu)-2-Chlorotritylresin was determined to be 0.88 mmol/g.

MeVal-Val-Dil-Dap-2-Chloro-Phe-2-Chlorotrityl Resin

MeVal-Val-Dil-Dap-2-Chloro-Phe-2-Chlorotrityl Resin was preparedfollowing General Procedure SP(c). Briefly, a 20% piperidine in DMFsolution (5-mL) was added to the syringe containingFmoc-2-Chloro-Phe-2-Chlorotrityl Resin, and the mixture was agitated for2 hours. The resin was filtered, washed with DMF (4×5 mL), DCM (4×5 mL),DMF (4×5 mL), DCM (4×5 mL) and ethyl ether (4×5 mL), and was driedin-vacuo for 2 h.

Fmoc-Dap (278 mg, 0.680 mmol) and HATU (259 mg, 0.680 mmol, 2 equiv.)were dissolved in anhydrous DMF (5-mL) and DIEA (237 μL, 1.36 mmol, 4equiv.). The resulting solution was transferred to the 10-mL plasticsyringe containing H-2-Chloro-Phe-2-Chlorotrityl Resin (555.6 mg, 0.340mmol). The mixture was agitated overnight at room temperature. Reactioncompletion was determined by the Kaiser amine test and LCMS analysis ofmaterial cleaved off a small amount of resin (using 2% TFA/CH₂Cl₂). Theresin was filtered, washed with DMF (4×5 mL), DCM (4×5 mL), DMF (4×5mL), DCM (4×5 mL) and ethyl ether (4×5 mL), and was dried in-vacuo for 2hours.

A 20% piperidine in DMF solution (5-mL) was added to the syringecontaining Fmoc-Dap-2-Chloro-Phe-2-Chlorotrityl Resin, and the mixturewas agitated for 2 hours. The resin was filtered, washed with DMF (4×5mL), DCM (4×5 mL), DMF (4×5 mL), DCM (4×5 mL) and ethyl ether (4×5 mL),and was dried in-vacuo for 2 hours.

Fmoc-MeVal-Val-Dil-OH (510 mg, 0.680 mmol, 2 equiv.) and HATU (259 mg,0.680 mmol, 2 equiv.) were dissolved in anhydrous DMF (5-mL) and DIEA(237 μL, 1.70 mmol, 5 equiv.). The resulting solution was transferred tothe 10-mL plastic syringe containing H-Dap-2-Chloro-Phe-2-Chlorotritylresin. The mixture was agitated for 6 hours. Reaction completion wasdetermined by LCMS analysis of material cleaved off a small amount ofresin (using 2% TFA/CH₂Cl₂). The resin was filtered, washed with DMF(4×5 mL), DCM (4×5 mL), DMF (4×5 mL), DCM (4×5 mL) and ethyl ether (4×5mL), and was dried in-vacuo for 2 hours.

MeVal-Val-Dil-Dap-2-Chloro-Phe-OH(SP2-a)

MeVal-Val-Dil-Dap-2-Chloro-Phe was prepared following General ProcedureSP(d). Briefly, a 20% piperidine in DMF solution (5-mL) was added to thesyringe containing Fmoc-MeVal-Val-Dil-Dap-2-Chloro-Phe-2-Chlorotritylresin, and the mixture was agitated for 2 hours. The resin was filtered,washed with DMF (4×5 mL), DCM (4×5 mL), DMF (4×5 mL), DCM (4×5 mL) andethyl ether (4×5 mL), and was dried in-vacuo for 2 hours. Further dryingwas achieved by leaving resin overnight under vacuum.

A 2% TFA/CH₂Cl₂ (5 mL) solution was transferred to a 10-mL plasticsyringe containing MeVal-Val-Dil-Dap-2-Chloro-Phe-2-Chlorotrityl resinand the mixture was agitated at room temperature for 5 minutes. Thefiltrate was collected in a 100 mL round-bottom flask. The process wasrepeated three times. The filtrate was evaporated to leave a whitesolid. Preparative HPLC purification provided 200 mg (67% TFA salt) ofwhite solid. Reversed-phase HPLC analysis: 96% at 6.72 mins. LC-MS m/z(ES⁺) calculated for C₃₉H₆₄ClN₅O₈, 765.44. found 767.063 (M+H)⁺.

Fmoc-MeVal-Val-Dil-Dap-Me-Phe-2-Chlorotrityl Resin

Fmoc-Dap-OH and Fmoc-MeVal-Val-Dil-OH were coupled, respectively, ontoH-Me-L-Phe-2-Chlorotrityl resin as described in General Procedure SP(c).

MeVal-Val-Dil-Dap-Me-Phe-OH(SP2-b)

MeVal-Val-Dil-Dap-Me-Phe-OH was cleaved off the resin as described inGeneral Procedure SP(d). The filtrate was evaporated to leave a whitesolid. Preparative HPLC purification provided 62.3 mg (26% TFA salt) ofwhite solid. Reversed-phase HPLC analysis: 98% at 6.88 mins. LC-MS m/z(ES⁺) calculated for C₄₀H₆₇N₅O₈, 745.5. found 746.908 (M+H)⁺.

Fmoc-MeVal-Val-Dil-Dap-Tic-2-Chlorotrityl Resin

Fmoc-Dap-OH and Fmoc-MeVal-Val-Dil-OH were coupled, respectively, ontoH-Tic-2-Chlorotrityl resin as described in General Procedure SP(c).

MeVal-Val-Dil-Dap-Tic-OH(SP2-c)

MeVal-Val-Dil-Dap-Tic-OH was cleaved off the resin as shown GeneralProcedure SP(d). The filtrate was evaporated to leave white solid.Preparative HPLC purification provided 178.40 mg (55% TFA salt) of whitesolid. Reversed-phase HPLC analysis: 98% at 6.74 mins. LC-MS m/z (ES⁺)calculated for C₄₀H₆₅N₅O₈, 743.48. found, 744.839 (M+H)⁺.

Fmoc-MeVal-Val-Dil-Dap-L-β-homophe-2-Chlorotrityl Resin

Fmoc-Dap-OH and Fmoc-MeVal-Val-Dil-OH were coupled, respectively, ontoH-L-β-homophe-2-Chlorotrityl Resin as described in General ProcedureSP(c).

MeVal-Val-Dil-Dap-L-β-homophe-OH(SP2-d)

MeVal-Val-Dil-Dap-L-β-homophe-OH was cleaved off the resin as describedin General Procedure SP(d). The filtrate was evaporated to leave whitesolid. Preparative HPLC purification provided 282.9 mg (99% TFA salt) ofwhite solid. Reversed-phase HPLC analysis: 98% at 6.65 mins. LC-MS m/z(ES⁺) calculated for C₄₀H₆₇N₅O₈ 745.5. found, 746.869 (M+H)⁺.

Fmoc-MeVal-Val-Dil-Dap-Boc-p-Amino-Phe-2-Chlorotrityl Resin

Fmoc-Dap-OH and Fmoc-MeVal-Val-Dil-OH were coupled, respectively, ontoBoc-p-Amino-Phe-2-Chlorotrityl resin as described in General ProcedureSP(c).

MeVal-Val-Dil-Dap-Boc-p-Amino-Phe-OH(SP2-e)

MeVal-Val-Dil-Dap-Boc-p-Amino-Phe-OH was cleaved off the resin asdescribed in General Procedure SP(d). The filtrate was evaporated toleave white solid. Preparative HPLC purification provided 210.6 mg (48%TFA salt) of white solid. Reversed-phase HPLC analysis: 98% at 6.9 mins.LC-MS m/z (ES⁺) calculated for C₄₄H₇₄N₆O₁₀, 846.55. found, 847.459(M+H)⁺.

Fmoc-MeVal-Val-Dil-Dap-3-cyclohexyl-L-Ala-2-Chlorotrityl Resin

Fmoc-Dap-OH and Fmoc-MeVal-Val-Dil-OH were coupled, respectively, ontoH-3-Cyclohexyl-L-Ala-2-Chlorotrityl resin as described in GeneralProcedure SP(c).

MeVal-Val-Dil-Dap-3-cyclohexyl-L-Ala-OH(SP2-f)

MeVal-Val-Dil-Dap-3-cyclohexyl-L-Ala-OH was cleaved off the resin asdescribed in General Procedure SP(d). The filtrate was evaporated toleave a white solid. Preparative HPLC purification provided 343.4 mg(99% TFA salt) of white solid. Reversed-phase HPLC analysis: 98% at 6.87mins. LC-MS m/z (ES⁺) calculated for C₃₉H₇₁N₅O₈, 737.53. found, 738.974(M+H)⁺.

Fmoc-MeVal-Val-Dil-Dap-L-4-Thiazolylalanine-2-Chlorotrityl Resin

Fmoc-Dap-OH and Fmoc-MeVal-Val-Dil-OH were coupled, respectively, ontoH-L-4-Thiazolylalanine-2-Chlorotrityl resin as described in GeneralProcedure SP(c).

MeVal-Val-Dil-Dap-L-4-Thiazolylalanine (SP2-g)

MeVal-Val-Dil-Dap-L-4-Thiazolylalanine was cleaved off the resin asdescribed in General Procedure SP(d). The filtrate was evaporated toleave a white solid. Preparative HPLC purification provided 357 mg (87%TFA salt) of a white solid. Reversed-phase HPLC analysis: 98% at 6.23mins. LC-MS m/z (ES⁺) calculated for C₃₉H₆₂N₆O₈S, 738.43. found, 739.889(M+H)⁺.

Fmoc-MeVal-Val-Dil-Dap-3-(3-pyridyl)-L-Ala-2-Chlorotrityl Resin

Fmoc-Dap-OH and Fmoc-MeVal-Val-Dil-OH were coupled, respectively, ontoH-3-(3-pyridyl)-L-Ala-2-Chlorotrityl resin as described in GeneralProcedure SP(c).

MeVal-Val-Dil-Dap-3-(3-pyridyl)-L-Ala-OH(SP2-h)

MeVal-Val-Dil-Dap-3-(3-pyridyl)-L-Ala-OH was cleaved off the resin asdescribed in General Procedure SP(d). The filtrate was evaporated toleave a white solid. Preparative HPLC purification provided 388.6 mg(94% TFA salt) of a white solid. Reversed-phase HPLC analysis: 98% at6.13 mins. LC-MS m/z (ES⁺) calculated for C₃₈H₆₄N₆O₈, 732.48. found,733.842 (M+H)⁺.

Fmoc-MeVal-Val-Dil-Dap-Leu-2-Chlorotrityl Resin

Fmoc-Dap-OH and Fmoc-MeVal-Val-Dil-OH were coupled, respectively, ontoH-Leu-2-Chlorotrityl resin as described in General Procedure SP(c).

MeVal-Val-Dil-Dap-Leu-OH(SP2-i)

MeVal-Val-Dil-Dap-Leu-OH was cleaved off the resin as described inGeneral Procedure SP(d). The filtrate was evaporated to leave a whitesolid. Preparative HPLC purification provided 217.4 mg (62% TFA salt) ofa white solid. Reversed-phase HPLC analysis: 98% at 6.43 mins. LC-MS m/z(ES⁺) calculated for C₃₆H₆₇N₆O₈, 697.5. found 698.999 (M+H)⁺.

Fmoc-MeVal-Val-Dil-Dap-Met-2-Chlorotrityl Resin

Fmoc-Dap-OH and Fmoc-MeVal-Val-Dil-OH were coupled, respectively, ontoH-Met-2-Chlorotrityl resin as described in General Procedure SP(c).

MeVal-Val-Dil-Dap-Met-OH(SP2-j)

MeVal-Val-Dil-Dap-Met-OH was cleaved off the resin as shown GeneralProcedure SP(d). The filtrate was evaporated to leave a white solid.Preparative HPLC purification provided 90.7 mg (82% TFA salt) of a whitesolid. Reversed-phase HPLC analysis: 98% at 6.39 mins. LC-MS m/z (ES⁺)calculated for C₃₅H₆₅N₅O₈S, 715.46. found 716.399 (M+H)⁺.

Fmoc-MeVal-Val-Dil-Dap-Trp(Boc)-2-Chlorotrityl Resin

Fmoc-Dap-OH and Fmoc-MeVal-Val-Dil-OH were coupled, respectively, ontoH-Trp-(Boc)-2-Chlorotrityl resin as described in General ProcedureSP(c).

MeVal-Val-Dil-Dap-Trp(Boc)-OH(SP2-k)

MeVal-Val-Dil-Dap-Trp(Boc)-OH was cleaved off the resin as described inGeneral Procedure SP(d). The filtrate was evaporated to leave a whitesolid. Preparative HPLC purification provided 151.7 mg (42% TFA salt) ofa white solid. Reversed-phase HPLC analysis: 98% at 7.39 mins. LC-MS m/z(ES⁺) calculated for C₄₆H₇₄N₆O₁₀, 870.55. found 871.645 (M+H)⁺.

Fmoc-MeVal-Val-Dil-Dap-Glu(OtBu)-2-Chlorotrityl Resin

Fmoc-Dap-OH and Fmoc-MeVal-Val-Dil-OH were coupled, respectively, ontoH-Glu(OtBu)-2-Chlorotrityl resin as described in General ProcedureSP(c).

MeVal-Val-Dil-Dap-Glu(OtBu)-OH(SP2-l)

MeVal-Val-Dil-Dap-Glu(OtBu)-OH was cleaved off the resin as described inGeneral Procedure SP(d). The filtrate was evaporated to leave a whitesolid. Preparative HPLC purification provided 219.4 mg (55% TFA salt) ofwhite solid. Reversed-phase HPLC analysis: 98% at 6.67 mins. LC-MS m/z(ES⁺) calculated for C₃₉H₇₁N₅O₁₀, 769.52. found 770.989 (M+H)⁺.

Example 3 Preparation of MC-Val-Cit-PAB-MMAZ

Maleimidocaproyl-Val-Cit-PAB-MeVal-Val-Dil-Dap-2-Chloro-Phe-OH(SP3-a)

Maleimidocaproyl-Val-Cit-PAB-OCOpNP was attached toMeVal-Val-Dil-Dap-2-Chloro-Phe-OH as described in General Procedure H.Preparative HPLC purification provided 13.50 mg (14%) of white solid.Reversed-phase HPLC analysis: 96% at 7.23 mins. LC-MS m/z (ES⁺)calculated for C₆₈H₁₀₂ClN₁₁O₁₆, 1363.72. found 1364.766 (M+H)⁺.

Maleimidocaproyl-Val-Cit-PAB-MeVal-Val-Dil-Dap-Me-Phe-OH(SP3-b)

Maleimidocaproyl-Val-Cit-PAB-OCOpNP was attached toMeVal-Val-Dil-Dap-Me-Phe-OH as described in General Procedure H.Preparative HPLC purification provided 17.1 mg (13%) of white solid.Reversed-phase HPLC analysis: 96% at 7.24 mins. LC-MS m/z (ES⁺)calculated for C₆₉H₁₀₅N₁₁O₁₆, 1343.77. found, m/z 1344.835 (M+H)⁺.

Maleimidocaproyl-Val-Cit-PAB-MeVal-Val-Dil-Dap-Tic-OH(SP3-c)

Maleimidocaproyl-Val-Cit-PAB-OCOpNP was attached toMeVal-Val-Dil-Dap-Tic-OH as described in General Procedure H.Preparative HPLC purification provided 2.7 mg (2%) of white solid.Reversed-phase HPLC analysis: 95% at 7.21 mins. LC-MS m/z (ES⁺)calculated for C₆₉H₁₀₃N₁₁O₁₆, 1341.76. found, m/z 1342.844 (M+H)⁺.

Maleimidocaproyl-Val-Cit-PAB-MeVal-Val-Dil-Dap-L-β-homophe-OH(SP3-d)

Maleimidocaproyl-Val-Cit-PAB-OCOpNP was attached toMeVal-Val-Dil-Dap-L-β-homophe-OH as described in General Procedure H.Preparative HPLC purification provided 3.1 mg (1.5%) of white solid.Reversed-phase HPLC analysis: 95% at 7.26 mins. LC-MS m/z (ES⁺)calculated for C₆₉H₁₀₅N₁₁O₁₆, 1343.77. found, m/z 1344.788 (M+H)⁺.

Maleimidocaproyl-Val-Cit-PAB-MeVal-Val-Dil-Dap-p-Amino-Phe-OH(SP3-e)

Maleimidocaproyl-Val-Cit-PAB-OCOpNP was attached toMeVal-Val-Dil-Dap-Boc-p-Amino-Phe-OH as described in General ProcedureH. Preparative HPLC purification provided 4.4 mg (2.5%) of white solid.Reversed-phase HPLC analysis: 95% at 7.54 min. LCMS calculated forC₇₃H₁₁₂N₁₂O₁₈ (MH)+ 1444.82. found, m/z 1445.972. A 50% solution ofTFA/CH₂Cl₂ (1 mL) was transferred toMaleimidocaproyl-Val-Cit-PABC-MeVal-Val-Dil-Dap-Boc-p-Amino-Phe-OH (3.0mg, 0.00263 mmol). Deprotection of Boc group was complete after 3 hours.The solvent was removed to leave a white solid. Preparative HPLCpurification provided 2.3 mg (82%) of white solid. Reversed-phase HPLCanalysis: 96% at 7.54 mins. LC-MS m/z (ES⁺) calculated forC₆₈H₁₀₄N₁₂O₁₆, 1344.77. found, 1345.539 (M+H)⁺.

Maleimidocaproyl-Val-Cit-PAB-MeVal-Val-Dil-Dap-3-cyclohexyl-L-Ala-OH(SP3-f)

Maleimidocaproyl-Val-Cit-PAB-OCOpNP was attached toMeVal-Val-Dil-Dap-3-cyclohexyl-L-Ala-OH as described in GeneralProcedure H. Preparative HPLC purification provided 1.5 mg (1%) of whitesolid. Reversed-phase HPLC analysis: 95% at 7.28 mins. LC-MS m/z (ES⁺)calculated for C₆₈H₁₀₉N₁₁O₁₆, 1336.66. found, 1337.166 (M+H)⁺.

Maleimidocaproyl-Val-Cit-PAB-MeVal-Val-Dil-Dap-L-4-Thiazolylalanine(SP3-g)

Maleimidocaproyl-Val-Cit-PAB-OCOpNP was attached toMeVal-Val-Dil-Dap-L-4-Thiazolyalanine as described in General ProcedureH. Preparative HPLC purification provided 0.5 mg (0.2%) of white solid.Reversed-phase HPLC analysis: 96% at 6.91 mins. LC-MS m/z (ES⁺)calculated for C₆₅H₁₀₉N₁₂O₆S, 1336.71. found, 1337.867 (M+H)⁺.

Maleimidocaproyl-Val-Cit-PAB-MeVal-Val-Dil-Dap-pyridyl)-L-Ala-OH(SP3-h)

Maleimidocaproyl-Val-Cit-PAB-OCOpNP was attached toMeVal-Val-Dil-Dap-3-(3-pyridyl)-L-Ala-OH as described in GeneralProcedure H. Preparative HPLC purification provided 4.4 mg (1.6%) ofwhite solid. Reversed-phase HPLC analysis: 98% at 6.94 mins. LC-MS m/z(ES⁺) calculated for C₆₇H₁₀₂N₁₂O₁₆, 1330.75. found, 1331.682 (M+H)⁺.

Maleimidocaproyl-Val-Cit-PAB-MeVal-Val-Dil-Dap-Leu-OH(SP3-i)

Maleimidocaproyl-Val-Cit-PAB-OCOpNP was attached toMeVal-Val-Dil-Dap-Leu-OH as described in General Procedure H.Preparative HPLC purification provided 10.3 mg (4.1%) of white solid.Reversed-phase HPLC analysis: 98% at 7.16 mins. LC-MS m/z (ES⁺)calculated for C₆₅H₁₀₅N₁₁O₁₆, 1295.77. found, 1296.524 (M+H)⁺.

Maleimidocaproyl-Val-Cit-PAB-MeVal-Val-Dil-Dap-Met-OH(SP3-j)

Maleimidocaproyl-Val-Cit-PAB-OCOpNP was attached toMeVal-Val-Dil-Dap-Met-OH as described in General Procedure H.Preparative HPLC purification provided 7.2 mg (6%) of white solid.Reversed-phase HPLC analysis: 98% at 7.06 mins. LC-MS m/z (ES⁺)calculated for C₆₄H₁₀₃N₁₁O₁₆S, 1313.73. found 1314.729 (M+H)⁺.

Maleimidocaproyl-Val-Cit-PAB-MeVal-Val-Dil-Dap-Trp(Boc)-OH(SP3-k)

Maleimidocaproyl-Val-Cit-PAB-OCOpNP was attached toMeVal-Val-Dil-Dap-Trp(Boc)-OH as described in General Procedure H.Preparative HPLC purification provided 7.4 mg (12%) of white solid.Reversed-phase HPLC analysis: 98% at 7.62 mins. LC-MS m/z (ES⁺)calculated for C₇₅H₁₁₂N₁₂O₁₈, 1468.82. found 1469.471 (M+H)⁺.

Maleimidocaproyl-Val-Cit-PAB-MeVal-Val-Dil-Dap-Glu(OtBu)-OH(SP3-l)

Maleimidocaproyl-Val-Cit-PAB-OCOpNP was attached toMeVal-Val-Dil-Dap-Glu(OtBu)-OH as described in General Procedure H.Preparative HPLC purification provided 2.9 mg (1.6%) of white solid.Reversed-phase HPLC analysis: 95% at 7.47 mins. LC-MS m/z (ES⁺)calculated for C₆₈H₁₀₉N₁₁O₁₈, 1367.8. found 1368.452 (M+H)⁺.

Example 4 Solution Phase Preparation of MMAZ (1)

The synthesis of MMAZ is described in Schemes 5 and 6. Fmoc-protectedamino acids can be prepared from unprotected amino acids using, forexample, Fmoc-OSu via well established procedures (see, e.g., Greene andWuts, Protective Groups in Organic Synthesis, 2nd Edition, 1991, JohnWiley & Sons, p. 506).

Preparation of Fmoc-Dolaproine (Fmoc-Dap)

Boc-Dolaproine (58.8 g, 0.205 mol) was suspended in 4 N HCl in1,4-dioxane (256 mL, 1.02 mol, Aldrich). After stirring for 1.5 hours,TLC analysis indicated the reaction was complete (10% MeOH/CH₂Cl₂) andthe mixture was concentrated to near-dryness. Additional 1,4-dioxane wascharged (50 mL) and the mixture was concentrated to dryness and driedunder vacuum overnight. The resulting white solid was dissolved in H₂O(400 mL) and transferred to a 3-L, three-neck, round-bottom flask with amechanical stirrer and temperature probe. N,N-diisopropylethylamine(214.3 mL, 1.23 mol, Acros) was added over one minute, causing anexotherm from 20.5 to 28.2° C. (internal). The mixture was cooled in anice bath and 1,4-dioxane was added (400 mL). A solution of Fmoc-OSu(89.90 g, 0.267 mol, Advanced ChemTech) in 1,4-dioxane (400 mL) wasadded from an addition funnel over 15 minutes, maintaining the reactiontemperature below 9° C. The mixture was allowed to warm to roomtemperature and stirred for 19 hours, after which the mixture wasconcentrated by rotary evaporation to an aqueous slurry (390 g). Thesuspension was diluted with H₂O (750 mL) and Et₂O (750 mL). The layerswere separated, keeping any solids with the organic layer. The aqueouslayer was acidified using conc. HCl (30 mL) and extracted with EtOAc(3×500 mL). The combined extracts were dried over MgSO₄, filtered andconcentrated. The Et₂O extract was extracted once with sat. NaHCO₃ (200mL), keeping any solids with the aqueous layer. The aqueous suspensionwas acidified using conc. HCl (50 mL) and extracted with Et₂O (50 mL),keeping any solids with the organic layer. The organic layer wasfiltered and concentrated. The two products were combined and purifiedby flash chromatography on silica gel eluting with CH₂Cl₂ (3.5 L), then3% MeOH/CH₂Cl₂ (9 L) to give 68.23 g of Fmoc-dolaproine as a white foam(81%, 97.5% purity by HPLC (AUC)).

Preparation of Fmoc-Dap-Z

The salt and/or protected form of the phenylalanine bioisostere (3mmol), N-Boc-Dolaproine (668 mg, 1 eq.), DEPC (820 μL, 1.5 eq.), andDIEA (1.2 mL) is diluted with dichloromethane (3 mL). After 2 hours (h)at room temperature (about 28 degrees Celsius), the reaction mixture isdiluted with dichloromethane (20 mL), washed successively with saturatedaqueous (aq.) NaHCO₃ (2×10 mL) and saturated aq. NaCl (2×10 mL). Theorganic layer is separated and concentrated. The resulting residue isre-suspended in ethyl acetate and is purified via flash chromatographyin ethyl acetate. The relevant fractions are combined and concentratedto provide the dipeptide. Protecting groups are cleaved by methods knownto those of skill in the art

Alternative Preparation of Fmoc-Dap-Z

Carboxy group protected Aminoacid Z (48.3 mmol) is suspended inanhydrous DMF (105 mL, Acros) for 5 minutes and Fmoc-Dap (19.80 g, 48.3mmol) is added. The mixture is cooled in an ice bath and TBTU (17.08 g,53.20 mmol, Matrix Innovations) is added. N,N-diisopropylethylamine(25.3 mL, 145.0 mmol, Acros) is added via syringe over 3 min. After 1hour, the ice bath is removed and the mixture is allowed to warm over 30min. The mixture is poured into water (1 L) and extracted with ethylacetate (300 mL). After separation, the aqueous layer is re-extractedwith ethyl acetate (2×150 mL). The combined organic layers are washedwith brine (150 mL), dried (MgSO₄) and filtered (filter paper) to removethe insolubles (inorganics and some dibenzofulvene). Afterconcentration, the residue is adsorbed on silica (41 g) and purified bychromatography (22 cm×8 cm column; 65% Heptane/EtOAc (2.5 L); 33%Heptane/EtOAc (3.8 L), to give product.

Preparation of Dap-Z

A 1-L round bottom flask is charged with Fmoc-Dap-Z, CH₂Cl₂ (122 mL) anddiethylamine (61 mL, Acros). The solution is stirred at room temperatureand the completion monitored by HPLC. After 7 hours, the mixture isconcentrated (bath temp. <30° C.). The residue is suspended in CH₂Cl₂(300 mL) and concentrated. This is repeated twice. To the residue isadded MeOH (20 mL) and CH₂Cl₂ (300 mL), and the solution isconcentrated. The residue is suspended in CH₂Cl₂ (100 mL) and toluene(400 mL), concentrated, and the residue left under vacuum overnight togive product.

Preparation of Fmoc-Meval-Val-Dil-Dap-Z

The tripeptide Fmoc-Meval-val-dil-O-t-Bu (prepared as described in WO02/088172, entitled “Pentapeptide Compounds and Uses Related Thereto”;0.73 mmol) is treated with TFA (3 mL) and dichloromethane (3 mL) for 2hours at room temperature. The mixture is concentrated to dryness. Theresidue is co-evaporated with toluene (3×20 mL) and dried in vacuumovernight. The residue is diluted with dichloromethane (5 mL) and addedto the deprotected dipeptide (287 mg, 0.73 mmol), followed by DIEA (550μL, 4 eq.) and DEPC (201 μL, 1.1 eq.). After 2 hours at room temperaturethe reaction mixture is diluted with ethyl acetate (50 mL), washedsuccessively with 10% aq. citric acid (2×20 mL), saturated aq. NaHCO₃(2×10 mL) and saturated aq. NaCl (10 mL). The organic layer is separatedand concentrated. The resulting residue is re-suspended in ethyl acetateand is purified via flash chromatography in ethyl acetate. The relevantfractions are combined and concentrated to provideFmoc-Meval-val-dil-dap-Z.

Alternative Preparation of Fmoc-MeVal-Val-Dil-Dap-Z

Crude Dap-Z (39.1 mmol) is suspended in anhydrous DMF (135 mL, Acros)for 5 minutes and Fmoc-MeVal-Val-Dil-OH (24.94 g, 39.1 mmol, see Example2 for preparation) is added. The mixture is cooled in an ice bath andTBTU (13.81 g, 43.0 mmol, Matrix Innovations) is added.N,N-Diisopropylethylamine (20.5 mL, 117.3 mmol, Acros) is added viasyringe over 2 minutes. After 1 hour, the ice bath is removed and themixture is allowed to warm over 30 min. The mixture is poured into water(1.5 L) and diluted with ethyl acetate (480 mL). After standing for 15minutes, the layers were separated and the aqueous layer is extractedwith ethyl acetate (300 mL). The combined organic layers are washed withbrine (200 mL), dried (MgSO4) and filtered (filter paper) to removeinsolubles (inorganics and some dibenzofulvene). After concentration,the residue (49 g) is scraped from the flask and adsorbed on silica (49g) and purified by chromatography (15 cm×10 cm dia column; 2:1EtOAc/Heptane (3 L), EtOAc (5 L); 250 mL fractions) to giveFmoc-MeVal-Val-Dil-Dap-Z.

Preparation of Meval-Val-Dil-Dap-Z

The product (0.2 mmol) is diluted with dichloromethane (3 mL),diethylamine (1 mL). The reaction mixture is stirred overnight at roomtemperature. Solvents are removed to provide an oil that is purified byflash silica gel chromatography in a step gradient 0-10% MeOH indichloromethane to provide Compound 1.

Using the above procedure, the compounds of the following formula areprepared:

Example 5 Synthesis of MMAZ Compounds

This synthesis describes the preparation of MMAZ compounds wherein

3-Phenylserine is available from Aldrich.

Synthesis of DimethylValine-Val-Dil-Dap-Phenylserine

To a suspension of Fmoc-Dap (1.2 g, 2.93 mmoles) in anhydrous CH₂Cl₂ (10mL) was added N,N′-disuccinimidyl carbonate (901 mg, 1.2 eq) followed byDIEA (1.28 mL, 2.5 eq). The reaction mixture was allowed to stir at roomtemperature overnight. Additional amounts of N,N′-disuccinimidylcarbonate (901 mg, 1.2 eq) followed by DIEA (1.28 mL, 2.5 eq) werecharged and stirring was continued for 18 h more. The reaction mixturewas diluted with EtOAc; organic layer was washed with 0.1 M aq. HCltwice, then dried over MgSO₄, filtered and concentrated in vacuo. Silicagel column chromatography in a step gradient of MeOH from 0 to 5% inCH₂Cl₂ afforded 1.12 g (75% yield) Fmoc-Dap-OSu as off-white foam.

Fmoc-Dap-OSu (0.615 g, 1.21 mmol) was suspended in dry DMSO (6 mL).D,L-threo-3-phenyl serine (0.2 g, 1.1 mmol) was added, and the reactionmixture was stirred overnight at room temperature. Mixture was directlyloaded on prep RP-HPLC and the product was isolated in a linear gradientof MeCN from 10 to 90% in aqueous 0.1% TFA. ObtainedFmoc-Dap-Phenylserine, 280 mg (44% yield), was suspended in dry CH₂Cl₂(2 mL) and treated with dimethyl amine (2 mL) for 4 hours at roomtemperature. Volatiles were removed under reduced pressure. Residue wasco-evaporated with Et₃N/CH₂Cl₂ 3 times to remove as much diethylamine aspossible, then dried in vacuo overnight. Residue was extensivelytriturated with ether to remove DBF. Dap-Phenylserine was dried and usedwithout further purification.

DimethylVal-Val-Dil-COOH (130 mg, 0.3 mmol, 1 eq), N-hydroxysuccinimide(39 mg, 0.3 mmol, 1 eq) and DCC (93 mg, 1.5 eq) were suspended in dryCH₂Cl₂ (1.5 mL). To this, DMAP (1 mg, cat.) was added and the reactionmixture was stirred at room temperature overnight. Precipitate wasfiltered off. Thus prepared DimethylVal-Val-Dil-OSu was suspended inCH₂Cl₂ (2 mL) and the mixture was added to Dap-Phenylserine, followed byDMSO (4 mL) and DIEA (100 uL). Reaction was allowed to stir at roomtemperature overnight. Precipitate was filtered off. CH₂Cl₂ was replacedby DMSO and the product was isolated by preparative RP-HPLC (lineargradient of MeCN, 10 to 90% in aq. 0.005% TFA) as two diastereomers.Isomer A: 84 mg, white foam. LC-MS m/z (ES⁺) 762.67 (M+H)⁺ at 10.58 min.Isomer B: 62 mg white solid. LC-MS m/z (ES⁺) 762.54 (M+H)⁺ at 10.67 min.

Synthesis of N-MethylValine-Val-Dil-Dap-Phenylserine

MeVal-Val-Dil-Dap-Phenylserine can be prepared as described above usingFmoc-MeVal-Val-Dil tripeptide. Fmoc can be later cleaved off the finaldrug according to General Procedure E.

Appropriately protected 3-phenylserine can be subjected to oxidizingconditions, e.g., pyridinium chlorochromate (PCC)/pyridine (see, e.g.,Synthesis, 1982, 245, 881, review), in order to provide thecorresponding ketone. The ketone can be further converted to varioushydrazones (hydrazones, acyl hydrazones, semicarbazones,thiosemicarbazones, etc.) as described, for example, by Kaneko et al.Bioconjugate Chemistry, 1991, 2(3), 133-141. Alternatively hydroxylgroup of amino- and carboxylate-protected 3-phenylserine can be readilycondensed with various acids using DCC/DMAP chemistry to provide esters(Larock, R. C., Comprehensive Organic Transformations, Wiley-VCH, 1999,p. 1937).

Methylphosphonate ester of 3-phenylserine can be generated by reactingmethylphosphonic diimidazolide (from commercially availablemethylphosphonic dichloride, Aldrich) with protected 3-phenylserinefollowed by aqueous hydrolysis.

3-Phenylserine phosphate ester can be generated by similar procedurefrom phosphorus oxychloride (Aldrich). Chemistries similar to thedescribed above can be used for the preparation of various derivativesof serine and threonine.

MMAZ is prepared by using the above phenylalanine analog and conjugatingwith Fmoc-Meval-val-dil-O-t-Bu following the procedure of Example 4.

Example 6 Synthesis of MMAZ Compounds

This synthesis describes the preparation of MMAZ compounds wherein

Enantiomerically pure diamino acids shown below wherein Hal is a halogencan be conveniently prepared as described in Zhou et al. 1999,Tetrahedron: Asymmetry 10(5):855-862.

MMAZ is prepared by using the above phenylalanine analog and conjugatingwith Fmoc-Meval-val-dil-O-t-Bu following the procedure of Example 4.

Example 7 Synthesis of MMAZ Compounds

This synthesis describes the preparation of MMAZ compounds wherein:

3-Aryl-glutamic acid and other 3-substituted pyroglutamic and glutamicacids can be prepared as described in Tetrahedron 9(2):217-229 (2002),or Journal of Organic Chemistry 66(4):1339-1350 (2001).

MMAZ is prepared by using the above phenylalanine analog and conjugatingwith Fmoc-Meval-val-dil-O-t-Bu following the procedure of Example 4.

Example 8 Synthesis of MMAZ Compounds

This synthesis describes the preparation of MMAZ compounds wherein:

3-Phenylcysteine can be prepared as described in Lago et al., 1992,Journal of Organic Chemistry 57(12):3493-6.

MMAZ is prepared by using the above phenylalanine analog and conjugatingwith Fmoc-Meval-val-dil-O-t-Bu following the procedure of Example 4.

Example 9 Synthesis of MMAZ Compounds

This synthesis describes the preparation of MMAZ compounds wherein:

2-Bromo-phenylalanine can be synthesized as described in Righi et al.,1996, Tetrahedron Letters 37(38):6893-6896.

MMAZ is prepared by using the above phenylalanine analog and conjugatingwith Fmoc-Meval-val-dil-O-t-Bu following the procedure of Example 4.

Example 10 Synthesis of MMAZ Compounds

This synthesis describes the preparation of MMAZ compounds wherein:

Beta-alkoxy-amino acids above, where R=alkyl, cyclohexyl, phenyl,benzyl, etc., can be synthesized as described in Bulletin of theChemical Society of Japan, 1982, 55(9):3049-50.

MMAZ is prepared by using the above phenylalanine analog and conjugatingwith Fmoc-Meval-val-dil-O-t-Bu following the procedure of Example 4.

Example 11 Synthesis of MMAZ Compounds

This synthesis describes the preparation of MMAZ compounds wherein:

Azi

The phenylalanine analogs are synthesized as described below. Aziridines(Azi) (infra), wherein Z=PhCH₂O₂C; R=H or Me, and R¹=PhCH₂ or Me, werecleaved by alcohols, R²OH, wherein R²=Me, Me₂CH, EtCHMe, Me₃C,cyclohexyl, PhCH₂, Ph, or the like, in the presence of BF₃.Et₂O toafford optically pure serine and threonine derivativesR₂OCHRCH(NHZ)CO₂R¹. The latter were deprotected by hydrogenolysis andsaponification to give the corresponding R₂OCHRCH(NH₂)CO₂H.

MMAZ is prepared by using the above phenylalanine analog and conjugatingwith Fmoc-Meval-val-dil-O-t-Bu following the procedure of Example 4.

Example 12 Synthesis of MMAZ Compounds

This synthesis describes the preparation of MMAZ compounds wherein:

Dehydrophenylalanine and other dehydro amino acids are synthesized asdescribed in Mathur et al., 2004, Biopolymers 76(2):150-161.

MMAZ is prepared by using the above phenylalanine analog and conjugatingwith Fmoc-Meval-val-dil-O-t-Bu following the procedure of Example 4.

Example 11 Synthesis of MMAZ Compounds

This synthesis describes the preparation of MMAZ compounds wherein:

β,β-dimethyl-phenylalanine and β,β-dimethyl-tyrosine can be synthesizedas described by Jonsson and Mikiver, 1976, Acta Pharmaceutica Suecica13(1):75-8. Refluxing PhCMe₂CH(CN)CO₂Et with N₂H₄ in MeOH gives 93% ofthe hydrazide with a pyrazolidine side product in 3.5% yield. Sequentialdiazotization, Curtius degradation, and hydrolysis give 74%PhCMe₂CH(NH₂)CO₂H. β,β-Dimethyltyrosine can be similarly prepared.

MMAZ is prepared by using the above phenylalanine analog and conjugatingwith Fmoc-Meval-val-dil-O-t-Bu following the procedure of Example 4.

Example 14 Synthesis of MMAZ Compounds

This synthesis describes the preparation of MMAZ compounds wherein:

4-(1-amino-2-phenylethyl)-benzoic acid can be prepared as described inJournal of Medicinal Chemistry 38(10):1600-7 (1995).

MMAZ is prepared by using the above phenylalanine analog and conjugatingwith Fmoc-Meval-val-dil-O-t-Bu following the procedure of Example 4.

Example 15 Synthesis of MMAZ Compounds

This synthesis describes the preparation of MMAZ compounds wherein:

The phenylalanine analogs are synthesized following the proceduresdescribed in Toth et al., 2004, Journal of the American Chemical Society126(34): 10538-10539.

MMAZ is prepared by using the above phenylalanine analog and conjugatingwith Fmoc-Meval-val-dil-O-t-Bu following the procedure of Example 4.

Example 16 Synthesis of MMAZ Compounds

This synthesis describes the preparation of MMAZ compounds wherein:

β,β-Difluoro analogs of α-oxo-β-phenylpropionic acid and phenylalanineare synthesized as shown below following the procedures described inSchlosser et al., 2004, Tetrahedron 60(35):7731-7742 and Roff et al.,2004, Journal of the American Chemical Society 126(13):4098-4099. Asimple three-step procedure converts the readily accessible(2-bromo-1,1-difluoroethyl)arenes into α-aryl-α,α-difluoroacetaldehydes.Subsequent hydrocyanation, hydrolysis, oxidation and further hydrolysisafforded β-aryl-β,β-difluoro-α-oxopropionic acids. Reductive aminationtransforms the oxo acids into a separable mixture of α-hydroxy acids andracemic β,β-difluoro-β-phenylalanine derivatives. Enantiomerically pureβ,β-difluorophenylalanine is obtained whenα,α-difluoro-α-phenylacetaldehyde is condensed with homochiral1-phenylethylamine, hydrogen cyanide is add to the resulting imine, thediastereomeric mixture thus produced is hydrolyzed to the carboxamideswhich is separable by fractional crystallization or chromatography.

Example 17 Synthesis of MMAZ Compounds

This synthesis describes the preparation of MMAZ compounds wherein:

A series of diastereoisomers ((2R,3S)-, (2S,3R)-, (2S,3S)- and (2R,3R))of β-methyl-β-arylalanine analogs can be prepared in enantiomericallypure form using a combination of chemo- and biocatalysis. Starting fromMe L-threoninate, a range of β,β-disubstituted didehydroamino acids areobtained as their (Z)-isomers. Asymmetric hydrogenation, using either[Rh(R,R)-Et-DuPhos(COD)]BF₄ or [Rh(S,S)-Et-DuPhos(COD)]BF₄ as acatalyst, followed by hydrolysis yielded the (2R,3S)- and (2S,3R)isomers, respectively. Subsequent enzymic stereoinversion of the (2R,3S)isomer with D-amino acid oxidase and stereoinversion of the (2S,3R)isomer with L-amino acid oxidase in combination with NH₃.BH₃ yields theremaining (2S,3S)- and (2R,3R) isomers, respectively.

Example 18 Synthesis of MMAZ Compounds

This synthesis describes the preparation of MMAZ compounds wherein:

Synthesis of 2-Amino-4-phosphonobutanoic acids

2-Amino-4-phosphonobutanoic Acids above, where Ar=phenyl, 3-pyridyl and2-thienyl, are synthesized as described in Ruiz et al., 2003, Journal ofOrganic Chemistry 68(20):7634-7645.

MMAZ is prepared by using the above phenylalanine analog and conjugatingwith Fmoc-Meval-val-dil-O-t-Bu following the procedure of Example 4.

Example 19 Synthesis of MMAZ of the Formula Above Wherein

Conjugate additions of lithiated bislactim ethers derived fromcyclo[Gly-Val] and cyclo[Ala-Val] to α-, β-, or α,β-substitutedvinylphosphonates allow direct and stereoselective access to a varietyof 3- or 4-monosubstituted and 2,3-, 2,4-, or 3,4-disubstituted2-amino-4-phosphonobutanoic acids (AP4 derivs.) in enantiomerically pureform. The relative stereochemistry can be assigned by x-ray diffractionanalysis or NMR study of 1,2-oxaphosphorinane derivs. Competitiveeight-membered “compact” and “relaxed” transition-state structures areinvoked to rationalize the stereochemical outcome of the conjugateadditions.

Example 20 Synthesis of MMAZ Compounds

This synthesis describes the preparation of MMAZ compounds wherein:

Synthesis of beta-substituted histidines is described in Wang et al.,2000, Tetrahedron Letters 41(9):1307-1310.

MMAZ is prepared by using the above phenylalanine analog and conjugatingwith Fmoc-Meval-val-dil-O-t-Bu following the procedure of Example 4.

Example 21 Synthesis of MMAZ Compounds

This synthesis describes the preparation of MMAZ compounds wherein:

Beta-fluoro amino acids are synthesized as described in Davis et al.,1999, Journal of Organic Chemistry 64(18):6931-6934.

MMAZ is prepared by using the above phenylalanine analog and conjugatingwith Fmoc-Meval-val-dil-O-t-Bu following the procedure of Example 4.

Example 22 Synthesis of MMAZ Compounds

This synthesis describes the preparation of MMAZ compounds wherein:

Beta-substituted glutamic acids, like the one above, can be prepared asdescribed in Ezquerra et al., 1999, Journal of Organic Chemistry64(18):6554-6565. The reaction of lithium enolates of achiralN-protected glycine esters with chiral alkoxyalkenylcarbene complexes ofchromium provide the corresponding Michael adducts with either high antior syn selectivity depending on the nature of the nitrogen protectinggroup, and high diastereofacial selectivity when carbene complexescontaining the (−)-8-phenylmenthyloxy group are employed. Subsequentoxidation of the metal-carbene moiety followed by deprotection of theamine group and hydrolysis of both carboxylic esters affordsenantiomerically enriched 3-substituted glutamic acids of natural aswell as unnatural stereochemistry. For example, carbene complex can bereacted with glycine lithium enolate to give the Michael additionadduct, which can be oxidized to give a protected glutamate without anyloss of stereochem. Glutamate is deprotected in two steps to give(2R,3S)-3-(3-furyl)glutamic acid hydrochloride salt. Alternatively, whenthe deprotection step is performed previous to the oxidation, cyclicaminocarbene complexes are formed, which will lead to optically active3-substituted pyroglutamic acids.

MMAZ is prepared by using the above phenylalanine analog and conjugatingwith Fmoc-Meval-val-dil-O-t-Bu following the procedure of Example 3.

Example 23 Synthesis of Other MMAZ Compounds

MMAZ compounds can also be prepared using the following commerciallyavailable phenylalanine analogs either as their protected or unprotectedamino acids incorporated in solution or solid phase synthesis asdescribed above:

(commercially available from Tyger Scientific, Inc. Ewing, N.J.);

(commercially available from Acros Organics);

(commercially available from Advanced ChemTech);

(commercially available from Acros);

(commercially available from Advanced ChemTech);

(commercially available from Advanced ChemTech);

(commercially available from Pharmacore Products);

(commercially available from Fluka);

(commercially available from Peptech);

(commercially available from Bachem);

(commercially available from ChemStep);

(commercially available from Chem IMPX);

(commercially available from Advanced ChemTech);

(commercially available from Chemstep);

(commercially available from Aldrich);

(commercially available from Sigma);

(commercially available from Apollo Scientific Ltd.);

(commercially available from DSL Chemicals (Shanghai) Co., Ltd.);

(commercially available from Salor);

(commercially available from Synchem OHG);

(commercially available from Sequoia Research Products Ltd.);

(commercially available from MicroChemistry Building Blocks);

(commercially available from Lancanster);

(commercially available from Ambinter, Paris, France);

(commercially available from Biomol Research Labs);

(commercially available from AstaTech);

(commercially available from ChemBridge Screening Library);

(commercially available from LaboTest);

(commercially available from JRD Fluorochemicals);

(commercially available from Fluka);

(commercially available from Senn Chemicals AG);

(commercially available from Advanced ChemTech);

(commercially available from Tyger Scientific);

(commercially available from AMRI Fine Chemicals);

(commercially available from Synthetech);

(commercially available from Apin Chemicals);

(commercially available from BioCatalytics, Inc.);

(commercially available from AG Scientific);

(commercially available from Synthelec);

(commercially available from TimTec Stock Library);

(commercially available from CSPS);

(commercially available from Qventas);

(commercially available from Encyclopedia of Amino Acid Analogs andChiral Building Blocks);

(commercially available from Bachem);

(commercially available from Matrix Scientific);

(commercially available from TCI America);

(commercially available from Acros);

(commercially available from Organics);

(commercially available from ChemPacific);

(commercially available from Rare Chemicals GmbH);

(commercially available from AstaTech);

(commercially available from Austin);

(commercially available from Advanced Asymmetrics, Inc.);

(commercially available from Tocris Cookson Inc.);

(commercially available from Chem Service, Inc.); and

(commercially available from Synchem OHG, Germany).

Example 24 Synthesis of MMAZ Compounds

MMAZ Compounds can also be prepared using the following commerciallyavailable phenylalanine analogs either as their protected or unprotectedamino acids incorporated in solution or solid phase synthesis asdescribed above: 4-chloro-phenylalanine, 4-fluoro-phenylalanine,4-nitro-phenylalanine, N-α-methyl-phenylalanine, α-methyl-phenylalanine,glutamic acid, aspartic acid, tryptophane, isoleucine, leucine,methionine, tyrosine, glutamine, threonine, valine, asparagine,phenylglycine, O-benzyl-serine, O-t-butyl-serine, O-t-butyl-threonine,homophenylalanine, methionine-DL-sulfoxide, methionine-sulfone,α-aminobutyric acid, α-aminoisobutyric acid,4-amino-1-piperidine-4-carboxylic acid,4-amino-tetrahydropyran-4-carboxylic acid, aspartic acid,benzothiazol-2-yl-alanine, α-t-butyl-glycine, cyclohexylalanine,norleucine, norvaline, S-acetamidomethyl-penicillamine,β-3-piperidin-3-yl-alanine, piperidinyl-glycine, pyrrolidinyl-alanine,selenocysteine, tetrahydropyran-4-yl-glycine, O-benzyl-threonine,O-t-butyl-tyrosine, 3-(p-acetylphenyl)alanine, 3-phenylserine, and1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid.

Example 25 Synthesis of MC-MMAZ

Maleimidocaproyl-MeVal-Val-Dil-Dap-Z can be prepared following GeneralProcedure S.

Briefly, maleimidocaproic acid (30 mg, Molecular Biosciences) andanhydrous DMF (10 μl) in 10 mL glass flask under Ar (balloon) are cooledon dry ice for 5 min. To this mixture oxalyl chloride (1 mL) is addedwith a syringe. (A vigorous reaction and pressure increase occurred.)After 5 min, the mixture is allowed to warm up to room temperature andleft for 30 min with occasional manual stirring. Volatiles are removedon Rotavap, residue is co-evaporated with anhydrous CH₂Cl₂ (1 mL) anddried at vacuum pump overnight. Product is initially generated as whitesolid, progressively turning into off-white to brownish solid. ¹H-NMR inCDCl₃: 1.26-1.32 (2H, m), 1.51-1.59 (2H, m), 1.63-1.70 (2H, m), 2.82(2H, t), 3.46 (2H, t), 6.70 (2H, s) ppm. Hydrolyzed material can bedetected by triplet at 2.35 ppm. Product is used if the integral of thetriplet at 2.42 ppm does not exceed 20% of the triplet at 3.46 ppm.

Maleimidocaproyl chloride can be prepared as described above anddissolved in anhydrous CH₂Cl₂ (3 mL).

MMAZ (1 eq.) and diisopropylethylamine (˜4 eq.) are dissolved inanhydrous CH₂Cl₂ in a glass flask equipped with magnetic stir bar andrubber cap. The reaction mixture is cooled on the ice bath for 10 minand maleimidocaproyl chloride solution (˜1.1 eq.) is added via syringe.After 15 min on ice, the reaction mixture is allowed to warm up to roomtemperature and stirring is continued for 2 more hours. Solvent is thenremoved in vacuo. The residue is suspended in DMSO (0.5 mL). Water (100μl) is then added and after 0.5 h mixture is loaded on preparative HPLCcolumn for separation: C₁₂ Phenomenex Synergi MAX-RP column, 4μ, 250×10mm, 80 Å. Monitoring is performed at 215 nm. Product containingfractions are concentrated on Rotavap, co-evaporated with acetonitrile(2×5 mL), then with mixture of CH₂Cl₂ and hexane to provide finalmaterial.

Example 26 Synthesis of an Analog of MC-MMAZ

The synthesis of an analog of MC-MMAZ is shown below.

MeVal-Val-Dil-Dap-Z-PG (compound 1, 0.044 mmol) is suspended in DMF(0.250 mL). 4-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-benzoic acid (11 mg,0.049 mmol) and HATU (17 mg, 0.044 mmol) are added followed by DIEA(0.031 mL, 0.17 mmol). This reaction mixture is allowed to stir for 2.0hr. HPLC analysis indicates complete consumption of starting compound 1.The product is isolated via preparatory RP-HPLC, using a Phenomenex C₁₂Synergi Max-RP 80 Å Column (250×21.20 mm). The eluent is a lineargradient of 10% to 80% MeCN/0.05% TFA (aq) over 8 minutes, thenisocratic 80% MeCN/0.05% TFA (aq) for an additional 12 minutes.MB-MeVal-Val-Dil-Dap-Z-PG (0.0385 mmol) is suspended in CH₂Cl₂ (1 mL)and TFA (1 mL). The mixture is stirred for 2 hr, and then volatileorganics are evaporated under reduced pressure. Product(MB-MeVal-Val-Dil-Dap-Z) is purified by preparatory RP-HPLC, using aPhenomenex C₁₂ Synergi Max-RP 80 Å Column (250×21.20 mm). The eluent isa linear gradient of 10% to 80% MeCN/0.05% TFA (aq) over 8 minutes, thenisocratic 80% MeCN/0.05% TFA (aq) for an additional 12 minutes.

Example 27 Preparation of MC-val-cit-PAB-MMAZ (9)

Compound 1 (0.11 mmol), Compound AB (85 mg, 0.12 mmol, 1.1 eq.), andHOBt (2.8 mg, 21 μmol, 0.2 eq.) are taken up in dry DMF (1.5 mL) andpyridine (0.3 mL) while under argon. After 30 h, the reaction is foundto be essentially complete by HPLC. The mixture is evaporated, taken upin a minimal amount of DMSO and purified by prep-HPLC (C₁₂-RP column,5μ, 100 Å, linear gradient of MeCN in water (containing 0.1% TFA) 10 to100% in 40 min followed by 20 min at 100%, at a flow rate of 25 mL/min)to provide Compound 9.

Compound 8 (32 μmol) is suspended in methylene chloride (6 mL) followedby the addition of TFA (3 mL). The resulting solution is allowed tostand for 2 hours. The reaction mixture is concentrated in vacuo andpurified by prep-HPLC (C₁₂-RP column, 5μ, 100 Å, linear gradient of MeCNin water (containing 0.1% TFA) 10 to 100% in 40 min followed by 20 minat 100%, at a flow rate of 25 mL/min). The desired fractions areconcentrated to providemaleimidocaproyl-valine-citrulline-p-hydroxymethylaminobenzene-MMAZ(MC-val-cit-PAB-MMAZ) 9.

Example 28 Preparation of AC10-MC-MMAZ by Conjugation of Antibody andMC-MMAZ

Antibody (e.g., AC10 or 1F6), dissolved in 500 mM sodium borate and 500mM sodium chloride at pH 8.0, is treated with an excess of 100 mMdithiothreitol (DTT). After incubation at 37° C. for about 30 minutes,the buffer is exchanged by elution over Sephadex G25 resin and elutedwith PBS with 1 mM DTPA. The thiol/Ab value is checked by determiningthe reduced antibody concentration from the absorbance at 280 nm of thesolution and the thiol concentration by reaction with DTNB (Aldrich,Milwaukee, Wis.) and determination of the absorbance at 412 nm. Thereduced antibody dissolved in PBS is chilled on ice.

The drug linker reagent, maleimidocaproyl-monomethyl auristatin Z, i.e.MC-MMAZ, dissolved in DMSO, is diluted in acetonitrile and water atknown concentration, and added to the chilled reduced antibody in PBS.After about one hour, an excess of maleimide is added to quench thereaction and cap any unreacted antibody thiol groups. The reactionmixture is concentrated by centrifugal ultrafiltration andantibody-MC-MMAZ is purified and desalted by elution through G25 resinin PBS, filtered through 0.2 μm filters under sterile conditions, andfrozen for storage.

Example 29 Preparation of Antibody-MC-val-cit-PAB-MMAZ by Conjugation ofAntibody and MC-val-cit-PAB-MMAZ (SP3, 9)

Antibody-MC-val-cit-PAB-MMAZ (e.g., AC10-MC-val-cit-PAB-MMAZ or1F6-MC-val-cit-PAB-MMAZ) is prepared by conjugation of the antibody andMC-val-cit-PAB-MMAZ (9, SP3) following the procedure of Example 28.

Example 30 Preparation of MC-MeVal-Cit-PAB-MMAZ

To a room temperature suspension of Fmoc-MeVal-OH (3.03 g, 8.57 mmol)and N,N′-disuccimidyl carbonate (3.29 g, 12.86 mmol) in CH₂Cl₂ (80 mL)is added DIEA (4.48 mL, 25.71 mmol). This reaction mixture is allowed tostir for 3 hr, and then poured into a separation funnel where theorganic mixture is extracted with 0.1 M HCl (aq). The crude organicresidue is concentrated under reduced pressure, and the product isisolated by flash column chromatography on silica gel using a 20-100%ethyl acetate/hexanes linear gradient (e.g., a total of 2.18 g of pureFmoc-MeVal-OSu (4.80 mmoles, 56% yield) can be recovered).

To a room temperature suspension of Fmoc-MeVal-OSu (2.18 g, 4.84 mmol)in DME (13 mL) and THF (6.5 mL) is added a solution of L-citrulline(0.85 g, 4.84 mmol) and NaHCO₃ (0.41 g, 4.84 mmol) in H₂O (13 mL). Thesuspension is allowed to stir at room temperature for 16 hr, then it isextracted into tert-BuOH/CHCl₃/H₂O and acidified to pH=2-3 with 1 M HCl.The organic phase is separated, dried and concentrated under reducedpressure. The residue is triturated with diethyl ether resultingFmoc-MeVal-Cit-COOH (e.g., 2.01 g) which is used without furtherpurification.

The crude Fmoc-MeVal-Cit-COOH is suspended in 2:1 CH₂Cl₂/MeOH (100 mL),and to it is added p-aminobenzyl alcohol (0.97 g, 7.9 mmol) and EEDQ(1.95 g, 7.9 mmol). This suspension is allowed to stir for 125 hr, thenthe volatile organics are removed under reduced pressure, and theresidue is purified by flash column chromatography on silica gel using a10% MeOH/CH₂Cl₂. Pure Fmoc-MeVal-Cit-PAB-OH (e.g., 0.55 g, 0.896 mmol,18.5% yield) is recovered.

To a suspension of Fmoc-MeVal-Cit-PAB-OH (0.55 g, 0.896 mmol) in CH₂Cl₂(40 mL) is added STRATOSPHERES™ (piperizine-resin-bound) (>5 mmol/g, 150mg). After being stirred at room temperature for 16 hr the mixture isfiltered through celite (pre-washed with MeOH), and concentrated underreduced pressure. The residue is triturated with diethyl ether andhexanes. The resulting solid material, MeVal-Cit-PAB-OH, is suspended inCH₂Cl₂ (20 mL), and to it is added MC-OSu (0.28 g, 0.896 mmol), DIEA(0.17 mL, 0.99 mmol), and DMF (15 mL). This suspension is stirred for 16hr. If HPLC analysis of the reaction mixture indicates an incompletereaction, the suspension is concentrated under reduced pressure to avolume of 6 mL, then a 10% NaHCO₃ (aq) solution is added and thesuspension stirred for an additional 16 hr. The solvent is removed underreduced pressure, and the residue is purified by flash columnchromatography on silica gel using a 0-10% MeOH/CH₂Cl₂ gradient,resulting in MC-MeVal-Cit-PAB-OH (e.g., 42 mg (0.072 mmol, 8% yield)).

To a suspension of MC-MeVal-Cit-PAB-OH (2.37 g, 4.04 mmol) andbis(nitrophenyl)carbonate (2.59 g, 8.52 mmol) in CH₂Cl₂ (10 mL) is addedDIEA (1.06 mL, 6.06 mmol). This suspension is stirred for 5.5 hr,concentrated under reduced pressure and purified by trituration withdiethyl ether. MC-MeVal-Cit-PAB-OCO-pNP (147 mg, 0.196 mmol) issuspended in a 1:5 pyridine/DMF solution (3 mL), and to it is added HOBt(5 mg, 0.039 mmol), DIEA (0.17 mL, 0.978 mmol) and MMAZ (0.205 mmol).This reaction mixture is stirred for 16 hr at room temperature, and thenpurified by preparatory RP-HPLC (×3), using a Phenomenex C₁₂ SynergiMax-RP 80 Å Column (250×21.20 mm). The eluent is a linear gradient of10% to 90% MeCN/0.05% TFA (aq) over 30 minutes, then isocratic 90%MeCN/0.05% TFA (aq) for an additional 20 minutes. MC-MeVal-Cit-PAB-MMAZis obtained.

Example 31 Preparation of Succinimide Ester of suberyl-Val-Cit-PAB-MMAZ

Compound 1 (0.38 mmol), Fmoc-Val-Cit-PAB-pNP (436 mg, 0.57 mmol, 1.5eq.) were suspended in anhydrous pyridine, 5 mL. HOBt (10 mg, 0.076mmol, 0.2 eq.) is added followed by DIEA (199 μl, 1.14 mmol, 3 eq.). Thereaction mixture is sonicated for 10 min, and then stirred overnight atroom temperature. Pyridine is removed under reduced pressure, and theresidue is re-suspended in CH₂Cl₂. The mixture is separated by silicagel flash chromatography in a step gradient of MeOH, from 0 to 10%, inCH₂Cl₂. The product containing fractions are collected, concentrated anddried in vacuum overnight to give Fmoc-Val-Cit-PAB-MMAZ.

Fmoc-Val-Cit-PAB-MMAZ is suspended in CH₂Cl₂ (2 mL) diethylamine (2 mL)and DMF (2 mL). The mixture is stirred for 2 hrs at room temperature.The solvent is removed under reduced pressure. The residue isco-evaporated with pyridine (2 mL), then with toluene (2×5 mL), anddried in vacuum. Val-Cit-PAB-MMAZ is obtained.

All Val-Cit-PAB-MMAZ prepared from Fmoc-Val-Cit-PAB-MMAZ is suspended inpyridine (2 mL), and added to a solution of disuccinimidyl suberate (74mg, 0.2 mmol, 4 eq.), in pyridine (1 mL). The reaction mixture isstirred at room temperature. After 3 hrs ether (20 mL) is added. Theprecipitate is collected and washed with additional amount of ether. Thereddish solid is suspended in 30% MeOH/CH₂Cl₂ and filtered through a padof silica gel with 30% MeOH/CH₂Cl₂ as an eluent.

Example 32 Determination of Cytotoxicity of Selected Compounds

The cytotoxic activity of MMAZ and antibody-drug conjugates is evaluatedon the CD70+ positive cell lines, for example, Caki-1, renal cellcarcinoma; L428, Hodgkin's disease; U251, glioblastoma. To evaluate thecytotoxicity of compounds, cells can be seeded at approximately 5-10,000per well in 150 μl of culture medium, then treated with graded doses ofcompounds in quadruplicates at the initiation of assay. Cytotoxicityassays are usually carried out for 96 hours after addition of testcompounds. Fifty μl of resazurin dye may be added to each well duringthe last 4 to 6 hours of the incubation to assess viable cells at theend of culture. Dye reduction can be determined by fluorescencespectrometry using the excitation and emission wavelengths of 535 nm and590 nm, respectively. For analysis, the extent of resazurin reduction bythe treated cells can be compared to that of the untreated controlcells.

Example 33 General In Vitro Cytotoxicity Determination

To evaluate the cytotoxicity of conjugates, cells are seeded atapproximately 5-10,000 per well in 150 μl of culture medium and thentreated with graded doses of conjugates in quadruplicates at theinitiation of assay. Cytotoxicity assays are carried out for 96 hoursafter addition of test compounds. Fifty μl of the resazurin dye is addedto each well during the last 4 to 6 hours of the incubation to assessviable cells at the end of culture. Dye reduction is determined byfluorescence spectrometry using the excitation and emission wavelengthsof 535 nm and 590 nm, respectively. For analysis, the extent ofresazurin reduction by the treated cells is compared to that of theuntreated control cells.

Example 34 In Vitro Cell Proliferation Assay

Efficacy of ADC can be measured by a cell proliferation assay employingthe following protocol (Promega Corp. Technical Bulletin TB288; Mendozaet al., 2002, Cancer Res. 62:5485-5488):

1. An aliquot of 100 μl of cell culture containing about 10⁴ cells(e.g., SKBR-3, BT474, MCF7 or MDA-MB-468) in medium is deposited in eachwell of a 96-well, opaque-walled plate.

2. Control wells are prepared containing medium and without cells.

3. ADC is added to the experimental wells and incubated for 3-5 days.

4. The plates are equilibrated to room temperature for approximately 30minutes.

5. A volume of CellTiter-Glo Reagent equal to the volume of cell culturemedium present in each well is added.

6. The contents are mixed for 2 minutes on an orbital shaker to inducecell lysis.

7. The plate is incubated at room temperature for 10 minutes tostabilize the luminescence signal.

8. Luminescence is recorded and reported in graphs as RLU=relativeluminescence units.

Table 7 shows in vitro activity of h1F6-antibody-MMAZ(h1F6-mc-vc-PAB-MMAZ) conjugates against CD70+ (U251, L428 and Caki-1)cell lines. Conjugates contain approximately 4 drugs per antibody.

TABLE 7 IC₅₀ (ng/mL) of h1F6-MC-val-cit-PAB-MMAZ conjugates on CD70+cell lines Z U251 Caki-1 L428 (L-2-Chlorophenylalanine)

8 4.4 8 (L-Me-Phenylalanine)

8 6 6 (L-Tic)

20 28 Maximum inhibition = 12% @ 1000 ng/ml (L-beta- homophenylalanine)

11 9 33 (L-Met)

23 18 43 (L-Leu)

14 16 105 (3-Pyridyl-L-alanine)

16 16 12 (L-4-thiazolylalanine)

6 7 4 (L-Trp)

7.5 6 11 (3-Cyclohexyl-L-alanine)

8 10 105 (Glu(OtBu)

43 51 Maximum inhibition = 12% @ 141 ng/ml (p-aminophenylalanine)

No effect No effect Maximum inhibition = 10% @ 100 ng/ml Phenylalanine(MMAF) 10 7 8

Example 35 Plasma Clearance in Rat

Plasma clearance pharmacokinetics of antibody drug conjugates and totalantibody is studied in Sprague-Dawley rats (Charles River Laboratories,250-275 grams each). Animals are dosed by bolus tail vein injection (IVPush). Approximately 300 μl whole blood is collected through jugularcannula, or by tail stick, into lithium/heparin anticoagulant vessels ateach timepoint: 0 (predose), 10, and 30 minutes; 1, 2, 4, 8, 24 and 36hours; and 2, 3, 4, 7, 14, 21, and 28 days post dose. Total antibody ismeasured by ELISA-ECD/GxhuFc-HRP. Antibody drug conjugate is measured byELISA-MMAZ/ECD-Bio/SA-HRP.

Example 36 Plasma Clearance in Monkey

Plasma clearance pharmacokinetics of antibody drug conjugates and totalantibody can be studied in cynomolgus monkeys, using a similar procedureto that described above.

Example 37 Tumor Volume In Vivo Efficacy in Transgenic Explant Mice

Animals suitable for transgenic experiments can be obtained fromstandard commercial sources such as Taconic (Germantown, N.Y.). Manystrains are suitable, but FVB female mice are preferred because of theirhigher susceptibility to tumor formation. FVB males can be used formating and vasectomized CD.1 studs can be used to stimulatepseudopregnancy. Vasectomized mice can be obtained from any commercialsupplier. Founders can be bred with either FVB mice or with 129/BL6×FVBp53 heterozygous mice. The mice with heterozygosity at p53 allele can beused to potentially increase tumor formation. Some F1 tumors are ofmixed strain. Founder tumors can be FVB only.

Animals having tumors (allograft propagated from Fo5 mmtv transgenicmice) can be treated with a single or multiple dose by IV injection ofADC. Tumor volume can be assessed at various time points afterinjection.

Example 38 In Vivo Efficacy of mcMMAZ Antibody-Drug Conjugates

The efficacy of cAC10-mcMMAZ can be evaluated in Karpas-299 ALCLxenografts. Chimeric AC10-mcMMAZ with an average of 4 drug moieties perantibody (cAC10-mcF4) is used. Karpas-299 human ALCL cells are implantedsubcutaneously into immunodeficient C.B-17 SCID mice (5×10⁶ cells permouse). Tumor volumes are calculated using the formula (0.5×L×W²) whereL and W are the longer and shorter of two bi-directional measurements.

Efficacy of cBR96-mcMMAZ in L2987 NSCLC Xenografts:

cBR96 is a chimeric antibody that recognizes the Le^(Y) antigen. Toevaluate the in vivo efficacy of cBR96-mcMMAZ with 4 drugs per antibody(cBR96-mcF4) L2987 non-small cell lung cancer (NSCLC) tumor fragmentsare implanted into athymic nude mice. When the tumors averageapproximately 100 mm³ the mice are divided into 3 groups: untreated and2 therapy groups. The efficacy of the antibody drug conjugates isevaluated as described above.

ATCC Deposits

An ATCC deposit of monoclonal antibody S2C6 was made on May 25, 1999pursuant to the terms of the Budapest Treaty on the internationalrecognition of the deposit of microorganisms for purposes of patentprocedure. This ATCC deposit was given an accession number of PTA-110.

An ATCC deposit of murine monoclonal antibody AC10 was made on Apr. 26,2005 pursuant to the terms of the Budapest Treaty on the internationalrecognition of the deposit of microorganisms for purposes of patentprocedure. This ATCC deposit was given an accession number of PTA-6679.

An ATCC deposit of monoclonal antibody humanized AC10 was made on Aug.23, 2005 pursuant to the terms of the Budapest Treaty on theinternational recognition of the deposit of microorganisms for purposesof patent procedure. This ATCC deposit was given an accession number ofPTA-6951.

The ATCC is located at 10801 University Boulevard, Manassas, Va.20110-2209, USA. Any deposit is provided as a convenience to those ofskill in the art and is not an admission that a deposit is requiredunder 35 U.S.C. Section 112. That described herein is not to be limitedin scope by the antibody deposited, since the deposited embodiment isintended as a single illustration of certain aspects of the inventionand any antibody that is functionally equivalent is within the scope ofthis invention. The deposit of material herein does not constitute anadmission that the written description herein contained is inadequate toenable the practice of any aspect of the invention, including the bestmode thereof, nor is it to be construed as limiting the scope of theclaims to the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

The present invention is not to be limited in scope by the specificembodiments disclosed in the examples which are intended asillustrations of a few aspects of the invention and any embodiments thatare functionally equivalent are within the scope of this invention.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart and are intended to fall within the scope of the appended claims.

All references cited herein are incorporated by reference in theirentirety and for all purposes to the same extent as if each individualpublication or patent or patent application was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes.

1. A compound having the formula:

or a pharmaceutically acceptable salt thereof, wherein: R² is C₁-C₈alkyl; R³ is C₁-C₈ alkyl; R⁴ is C₁-C₈ alkyl; R⁵ is selected from thegroup consisting of H and methyl; R⁶ is C₁-C₈ alkyl; R⁷ is C₁-C₈ alkyl;each R⁸ is independently selected from O—(C₁-C₈ alkyl); and the moiety—NR⁹Z¹ is a phenylalanine bioisostere moiety selected from the groupconsisting of,


2. The compound of claim 1 or a pharmaceutically acceptable saltthereof, wherein R² is methyl.
 3. The compound of claim 1 or apharmaceutically acceptable salt thereof, wherein R³ is isopropyl. 4.The compound of claim 1 or a pharmaceutically acceptable salt thereof,wherein R⁴ is isopropyl.
 5. The compound of claim 1, wherein thephenylalanine bioisostere moiety is


6. A compound having the formula:L-((LU)-(D)₁₋₄)_(p) or a pharmaceutically acceptable salt thereofwherein, L- is a Ligand Unit selected from the group consisting of aprotein, polypeptide and peptide; LU is a Linker Unit; p is an integerof from 1 to about 20; and D is a drug moiety having Formula D:

wherein, R² is C₁-C₈ alkyl; R³ is C₁-C₈ alkyl; R⁴ is C₁-C₈ alkyl; R⁵ isselected from the group consisting of H and methyl; R⁶ is C₁-C₈ alkyl;R⁷ is C₁-C₈ alkyl; each R⁸ is independently selected from O—(C₁-C₈alkyl); and the moiety —NR⁹Z¹ is a phenylalanine bioisostere moietyselected from the group consisting of


7. A compound of claim 6, having the formula:L-[LU-D]_(p) or a pharmaceutically acceptable salt thereof.
 8. Thecompound according to claim 6, or a pharmaceutically acceptable saltthereof, wherein: L is an antibody (Ab), LU is a Linker Unit having theformula -A_(a)-W_(w)—Y_(y)—, wherein A is a Stretcher unit, a is 0or 1,each W is independently an Amino Acid unit, w is an integer ranging from0 to 12, Y is a Spacer unit, and y is 0, 1 or
 2. 9. The compound ofclaim 8 or a pharmaceutically acceptable salt thereof having theformula:


10. The compound of claim 8 having the formula:


11. The compound of claim 8 having the formula:


12. The compound of claim 8 or a pharmaceutically acceptable saltthereof wherein w is an integer ranging from 2 to
 12. 13. The compoundof claim 8 or a pharmaceutically acceptable salt thereof wherein w is 2.14. The compound of claim 13 or a pharmaceutically acceptable saltthereof, wherein W_(W) is -valine-citrulline-.
 15. The compound of claim8 wherein W_(W) is selected from the group consisting of 5-aminovalericacid, homophenylalanine-lysine, tetraisoquinolinecarboxylate-lysine,cyclohexylalanine-lysine, isonepecotic acid-lysine, beta-alanine-lysine,glycine-serine-valine-glutamine (SEQ ID NO:1) and isonepecotic acid. 16.The compound of claim 8 or a pharmaceutically acceptable salt thereof,wherein the antibody is a monoclonal antibody.
 17. The compound of claim8, wherein the antibody is an antibody fragment.
 18. The compound ofclaim 8 having the formulae:

or a pharmaceutically acceptable salt thereof wherein mAb is amonoclonal antibody and

is a phenylalanine bioisostere moiety selected from the group consistingof


19. The compound of claim 8 or a pharmaceutically acceptable saltthereof, wherein the antibody specifically binds to CD20, CD30, CD33,CD40, CD70, or Lewis Y antigen.
 20. A pharmaceutical compositioncomprising an effective amount of the compound of claim 6, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable diluent, carrier or excipient.
 21. The compound of claim 12or a pharmaceutically acceptable salt thereof wherein y is 1 or
 2. 22.The compound of claim 6, wherein the phenylalanine bioisostere moiety is


23. The compound of claim 8, wherein the phenylalanine bioisosteremoiety is


24. The compound of claim 18, wherein the phenylalanine bioisosteremoiety is


25. A compound having the formula: LU-(D)₁₋₄ or a pharmaceuticallyacceptable salt thereof wherein, LU- is a Linker Unit; and D is a drugmoiety having the Formula D:

wherein R² is C₁-C₈ alkyl; R³ is C₁-C₈ alkyl; R⁴ is C₁-C₈ alkyl; R⁵ isselected from the group consisting of H and methyl; R⁶ is C₁-C₈ alkyl;R⁷ is C₁-C₈ alkyl; each R⁸ is independently selected from O—(C₁-C₈alkyl); and the moiety —NR⁹Z¹ is a phenylalanine bioisostere selectedfrom the group consisting of


26. The compound of claim 25, wherein the phenylalanine bioisosteremoiety is